CN107881127B - Bacillus amyloliquefaciens Lxz-41 and method for controllably preparing nano-selenium by using same - Google Patents
Bacillus amyloliquefaciens Lxz-41 and method for controllably preparing nano-selenium by using same Download PDFInfo
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Abstract
The invention provides bacillus amyloliquefaciens Lxz-41 and a method for preparing nano-selenium controllably by using the strain. The preservation number of the bacillus amyloliquefaciens Lxz-41 is as follows: CCTCC M2016578, deposited in China center for type culture Collection, and has controllable production capacity of nano selenium. The invention takes bacillus amyloliquefaciens Lxz-41 as a fermentation strain, selenite is fed, the culture time, the rotating speed and the surfactant concentration of the strain are controlled to controllably prepare nano-selenium, and fermentation liquor is collected, precipitated and purified to obtain nano-selenium particles. The strain disclosed by the invention is resistant to high-concentration selenite, and the particle size of most of generated nano-selenium particles can be controlled below 200nm by controlling the preparation process conditions. The controllable nano-selenium preparation method can reduce the inhibition effect of selenate on the growth of thalli, increase the conversion rate of nano-selenium, effectively control the agglomeration phenomenon of nano-selenium particles, and has the advantages of simple required culture medium and culture conditions, low cost and easy operation, thereby being suitable for large-scale fermentation tank production.
Description
Technical Field
The invention belongs to the fields of microorganism application technology and nano-selenium preparation, and particularly relates to bacillus amyloliquefaciens Lxz-41 and a method for preparing nano-selenium controllably by using the strain.
Background
Selenium is a very important nutrient element in the ecosystem and is called as a 'vital element'. As early as 1957, Schwarz studies determined that selenium is an essential nutrient for humans and animals. In 1973, Rotruk et al discovered that selenium is an essential active component of glutathione peroxidase for higher animal biological metabolism. In the same year, selenium was recognized by the world health organization as a trace element essential to the human body. Since then, extensive studies have shown that selenium is very narrow in its nutritional range. Selenium deficiency can be caused when the selenium content in human food is less than 0.05mg/kg, and poisoning can be caused when the selenium content is more than 5 mg/kg. Selenium is unevenly distributed on the earth, and parts of high-selenium areas and serious selenium-deficiency areas appear. 72% of China is in selenium-deficient and low-selenium bands, and the deficiency of the intake of selenium in diet seriously affects the health of hundreds of millions of people. According to the survey report of Chinese Nutrition society, the daily intake of selenium for adults is only 26.63ug, which is far from the daily intake of 50ug recommended by the Chinese Nutrition society and the International society for selenium society. The selenium deficiency can cause keshan disease, Kashin-Bek disease, cancer, diabetes, cardiovascular and cerebrovascular diseases, infertility, etc., and the appropriate selenium supplement can prevent cancer, tumor, AIDS and aging. Selenium deficiency has seriously threatened the health of people and caused potential hazards. In recent years, with more and more attention paid to health care and health preservation, various selenium-rich products come into play, but the quality of the products is uneven, and the content of selenium does not have a unified standard. And because of the uneven selenium content in the soil, inorganic selenium (sodium selenate and sodium selenite) is sprayed on the selenium-rich product in the planting process to improve the selenium content in the product. However, inorganic selenium (sodium selenate and sodium selenite) has high toxicity and is classified as a sixth type of highly toxic substance, and the use of inorganic selenium seriously harms the environment and human health. Therefore, the current primary research task is to develop a high-performance and low-toxicity selenium source, which will be the key point of selenium nutrition health care research
Research on the preparation of selenium-containing compounds with high activity and low toxicity has begun, and the research on the aspect is relatively slow at present. Traditionally, two forms of selenium are included: one is selenium compound with both activity and toxicity, and the other is zero-valent selenium with low toxicity. Among them, zero-valent selenium mainly exists in three forms of gray, red and black, and for the three forms of elemental selenium, it is well known that the particle size of gray and black selenium is large and has no biological activity, while nanoscale red selenium has biological activity according to acute toxicity (LD)50) Data display, inorganic selenium LD5015mg/kg, organic selenium LD5030-40mg/kg, nano selenium LD50113 mg/kg. To date, nanoselenium has been found to be the least toxic. The existing production method of nano-selenium mainly comprises a chemical synthesis method andthe biological nano selenium is more uniform and more regular than the chemically synthesized nano selenium in the biological synthesis method, and the biological nano selenium is high temperature resistant and more stable and is not easy to be converted into black or brown nano selenium. The biosynthesis of nano-selenium is mainly produced by reducing selenite by microorganisms, and with the continuous research, the species covered by the microorganisms capable of synthesizing nano-selenium are extremely abundant, and more than 30 species comprise Escherichia coli (Escherichia coli), Rhodobacter capsulatus (Rhodobacter Imhoff), Bacillus subtilis (Bacillus subtilis) and the like. However, most microorganisms are not easy to ferment and produce and store, so that industrial production cannot be realized. The bacillus can produce spores with stronger stress resistance, can resist adverse environment and conditions in the production and processing process, and is easy to store, so that the bacillus is a good microbial strain for industrially producing nano-selenium. However, due to various factors, the produced microbial nano selenium particles have large particle size and are different in size.
Disclosure of Invention
The invention aims to provide bacillus amyloliquefaciens Lxz-41, which has the capability of controllably preparing nano selenium.
The technical scheme adopted by the invention is as follows:
a Bacillus amyloliquefaciens Lxz-41 with a preservation number of: CCTCC M2016578, deposited in China center for type culture Collection, and has controllable production capacity of nano selenium.
A method for controllably producing nano-selenium by Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Lxz-41 comprises the following steps:
s1, activating strains: inoculating the preserved strain to a beef extract peptone or LB test tube agar slant culture medium, and performing static culture and activation at 25-40 ℃ until the slant surface is full of bacterial colonies;
s2, seed culture: inoculating slant culture strain into beef extract peptone liquid culture medium containing selenium salt, loading liquid amount is 20% of container capacity, and performing shake culture at 25-40 deg.C and 80-200r/min for 12-24 hr to obtain seed liquid;
s3, controllable production of nano-selenium: inoculating the seed culture solution into a fermentation container filled with a fermentation culture medium according to the inoculation amount of 1-5%, wherein the fermentation culture medium comprises selenium salt, the selenium salt is added in a fed-batch manner, the liquid loading amount of the fermentation culture medium is 40-70% of that of the fermentation container, the stirring speed is 50-200r/min, the ventilation amount is 0.5-2.0vvm, the temperature is kept at 25-40 ℃, and the fermentation time is 24-72 h;
s4, extracting and purifying nano selenium: and (2) putting the fermentation liquid in a tank, performing refrigerated centrifugation at the temperature of 4000 plus 10000r/min4 ℃ for 10-20min, collecting the obtained thallus, washing the thallus with sterile normal saline with the volume of about 1/4 fermentation liquid for three times, suspending the thallus with sterile distilled water with the volume of about 1/20, performing ice bath ultrasonic wall breaking for 20-40min, performing refrigerated centrifugation at the temperature of 4 ℃ for 10-20min on thallus lysate 4000 plus 10000r/min, cleaning the precipitate with deionized water with the same volume for three times, suspending the precipitate with distilled water with the volume of about 1/2 to obtain nano-selenium suspension, adding chloroform with the same volume for extraction for 30min, extracting for 2-3 times, combining lower-layer water phases, homogenizing the precipitate with a homogenizer for 2-5min, performing refrigerated centrifugation at the temperature of 4000 plus 10000r/min4 ℃ for 10-20min, cleaning the precipitate with equal-volume sterile normal saline for 3 times, and performing refrigerated drying to obtain the microbial nano-selenium.
Furthermore, the selenium salt in the liquid culture medium of the beef extract peptone in S2 is one or more of selenite and selenate, and the mass-to-volume ratio is 100-500 mg/L.
Selenium salts in the S2 and S3 media were separately sterilized and then added to the sterilized media.
Further, the fermentation medium in S3 comprises the following components in percentage by weight (g/L): glucose 10, peptone 5, ammonium sulfate 1, magnesium sulfate heptahydrate 0.3, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate 0.5, sodium chloride 2, selenium salt 0.1-4.5, surfactant 0.5-1, and pH 7.2-7.4.
Further, selenium salt in the fermentation medium in the S3 is fed batch or continuously fed batch by mother liquor to control the concentration of the selenium salt in the fermentation liquid and further control the transformation efficiency of the strain, wherein the selenium salt is one or more of selenite and selenate.
Further, sodium selenite is fed in batch or continuously in the fermentation medium, and the concentration of the used sodium selenite mother liquor is 50-500 g/L.
Further, in S4, the ultrasonic crushing power is 400W-1000W, the frequency is 20-40KHz, the start-stop interval is 10-15S, and the crushing is 20-40 min.
Further, the surfactant is one or more of SDS, Tween 40, Tween 60 and Tween 80.
The invention has the following advantages:
1. the bacillus amyloliquefaciens Lxz-41 used in the invention can tolerate sodium selenite with the concentration of 5000mg/L which is higher than the tolerance concentration of bacillus reported before, and the invention adopts a mode of adding selenite in batches, low-concentration selenite is added in the adaptation period in the previous period according to the growth rule of thalli, and the concentration of selenite is gradually increased along with the growth of thalli. Therefore, the problem of toxicity to the thalli caused by one-time addition of selenite can be solved, and the concentration of the selenite can be always maintained under the optimal conversion concentration of the thalli under the condition of fed-batch, so that the conversion efficiency of the nano-selenium is improved.
2. The invention controls the particle size distribution of the obtained nano-selenium by controlling the stirring speed, the concentration of the surfactant and the fermentation time in the fermentation process. The larger the stirring speed is, the smaller the particle size is; in the range of not influencing the growth concentration of the thalli, the larger the concentration of the surfactant is, the smaller the particle size is; the shorter the fermentation time, the smaller the particle size, within the range not affecting the growth of the cells.
3. The bacillus amyloliquefaciens Lxz-41 selected by the invention can resist high-concentration sodium selenite, the strains belong to probiotics and are safe to use, the bacillus amyloliquefaciens metabolites are rich, and the secreted antibiotic antibacterial protein or polypeptide substances and the like are added into the fertilizer, so that better biological control and selenium enrichment effects can be achieved; can also be directly used as feed additive to be added into livestock and poultry feed. The bacillus amyloliquefaciens Lxz-41 has extensive culture conditions, is not easy to contaminate bacteria, has low cost, and the method for preparing the nano selenium by utilizing the modified strain has the advantages of simple operation, low cost, convenient preparation, high yield and the like, and is suitable for large-scale preparation.
Drawings
FIG. 1 is a bar graph comparing selenite concentration and nano-selenium conversion rate between fed-batch and one-time sodium selenite additions according to the present invention;
FIG. 2 is a bar graph comparing the selenite concentration and the thallus growth when sodium selenite is fed in and added at one time according to the present invention;
FIG. 3 is a bar graph comparing the average particle size change for the nano-selenium produced at different agitation rates according to the present invention;
FIG. 4 is a bar graph comparing the variation of average particle size for the nano-selenium produced at different fermentation times according to the present invention;
FIG. 5 is a bar graph comparing the change in average particle size for the nano-selenium produced at different surfactant concentrations according to the present invention;
FIG. 6 is a characteristic diagram of a strain of the present invention;
FIG. 7 shows the results of sodium selenite-tolerant concentration experiments with the strains of the present invention;
FIG. 8 is a photo of the production of nano-selenium by liquid shake flask fermentation of the strain of the present invention;
FIG. 9 is an electron microscope scanning photograph of nano-selenium prepared by the method of the present invention with a resolution of 1 μm;
FIG. 10 is an SEM image of the resolution of 100nm of nano-selenium prepared by the method of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. Those skilled in the art will readily recognize that certain features may be omitted from the examples or may be substituted with other elements, materials, methods. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.
Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.
In the present specification, the method for detecting the average particle size is as follows:
three groups of parallel nano selenium particles (three groups of nano selenium particles obtained under the same preparation condition) are selected for shooting, the particle size of the Ni nano selenium particles is randomly selected and measured in each picture, and the average particle size is calculated by the following formula.
The invention separates a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Lxz-41 capable of tolerating high-concentration sodium selenite from the purple positive soil of Ankang city, Shanxi province, wherein the strain Lxz-41 is currently stored in the China center for type culture Collection with the address: wuhan city Wuchang Luojia mountain, post code 430072, preservation number CCTCC M2016578, and preservation date 2016, 10 months and 18 days.
The 16srDNA sequence of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Lxz-41 is shown as follows:
AATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTA
the invention also provides a controllable method for preparing nano-selenium by utilizing Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Lxz-41, which comprises the following steps:
s1, activating strains: inoculating the preserved strain to a beef extract peptone or LB test tube agar slant culture medium, and performing static culture and activation at 25-40 ℃ until the slant surface is full of bacterial colonies;
s2, seed culture: inoculating slant culture strain into beef extract peptone liquid culture medium containing selenium salt, loading liquid amount is 20% of container capacity, and performing shake culture at 25-40 deg.C and 80-200r/min for 12-24 hr to obtain seed liquid;
s3, controllable production of nano-selenium: inoculating the seed culture solution into a fermentation container filled with a fermentation culture medium according to the inoculation amount of 1-5%, wherein the fermentation culture medium comprises selenium salt, the selenium salt is added in a fed-batch manner, the liquid loading amount of the fermentation culture medium is 40-70% of that of the fermentation container, the stirring speed is 50-200r/min, the ventilation amount is 0.5-2.0vvm, the temperature is kept at 25-40 ℃, and the fermentation time is 24-72 h;
s4, extracting and purifying nano selenium: and (2) putting the fermentation liquid in a tank, performing refrigerated centrifugation at the temperature of 4000 plus 10000r/min4 ℃ for 10-20min, collecting the obtained thallus, washing the thallus with sterile normal saline with the volume of about 1/4 fermentation liquid for three times, suspending the thallus with sterile distilled water with the volume of about 1/20, performing ice bath ultrasonic wall breaking for 20-40min, performing refrigerated centrifugation at the temperature of 4 ℃ for 10-20min on thallus lysate 4000 plus 10000r/min, cleaning the precipitate with deionized water with the same volume for three times, suspending the precipitate with distilled water with the volume of about 1/2 to obtain nano-selenium suspension, adding chloroform with the same volume for extraction for 30min, extracting for 2-3 times, combining lower-layer water phases, homogenizing the precipitate with a homogenizer for 2-5min, performing refrigerated centrifugation at the temperature of 4000 plus 10000r/min4 ℃ for 10-20min, cleaning the precipitate with equal-volume sterile normal saline for 3 times, and performing refrigerated drying to obtain the microbial nano-selenium.
Selenium salt in the liquid culture medium of the beef extract peptone in S2 is one or more of selenite and selenate, and the mass-to-volume ratio is 100-500 mg/L.
Selenium salts in the S2 and S3 media were separately sterilized and then added to the sterilized media.
The fermentation medium in S3 comprises the following components (g/L): glucose 10, peptone 5, ammonium sulfate 1, magnesium sulfate heptahydrate 0.3, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate 0.5, sodium chloride 2, selenium salt 0.1-4.5, surfactant 0.5-1, and pH 7.2-7.4.
And (3) selenium salt in the fermentation medium in the S3 is fed batch or continuously fed batch by mother liquor to control the concentration of the selenium salt in the fermentation liquid and further control the conversion efficiency of the strain, wherein the selenium salt is one or more of selenite and selenate.
Adding sodium selenite in batch or continuous manner in fermentation culture medium, wherein the concentration of sodium selenite mother liquor is 50-500 g/L.
In S4, the ultrasonic crushing power is 400W-1000W, the frequency is 20-40KHz, the start-stop interval is 10-15S, and the crushing is 20-40 min.
The surfactant is one or more of SDS, Tween 40, Tween 60 and Tween 80.
FIG. 7 shows the experimental results of the tolerance concentration of the strain of the present invention to sodium selenite, and it can be seen from the figure that Bacillus amyloliquefaciens can tolerate 5000mg/L of sodium selenite, the concentration range of the sodium selenite most suitable for growth of the bacteria is 1500mg/L-3000mg/L, and the tolerance capability of the strain to high sodium selenite improves the production capability of nano-selenium.
Referring to fig. 1 and 2, a bar graph comparing selenite concentration and nano-selenium conversion rate at 48h fermentation time and at one time seed culture liquid is shown; as can be seen from the figure, the selenite is fed in batch, low-concentration selenite is added in the adaptation period in the early stage according to the growth rule of the thalli, and the concentration of the selenite is gradually increased along with the growth of the thalli. Therefore, the problem of toxicity to the thalli caused by one-time addition of selenite can be solved, and the concentration of the selenite can be always maintained under the optimal conversion concentration of the thalli under the condition of fed-batch, so that the conversion efficiency of the nano-selenium is improved.
Referring to fig. 3-5, the size distribution of the obtained nano-selenium particle size is controlled by controlling the stirring rate, the surfactant concentration and the fermentation time during the fermentation process. The larger the stirring speed is, the smaller the particle size is; in the range of not influencing the growth concentration of the thalli, the larger the concentration of the surfactant is, the smaller the particle size is; the shorter the fermentation time, the smaller the particle size, within the range not affecting the growth of the cells.
Finally, a photo of the nano-selenium produced by liquid shake-flask fermentation of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Lxz-41 is shown in figure 8, sodium selenite is added at the concentration of 2000mg/L, 3000mg/L and 140r/min, and the solution is changed into bright red after being cultured for 24 hours at the temperature of 28 ℃; the microstructure of the nano-selenium particles obtained by collecting the fermentation liquid precipitate and purifying is shown in fig. 9 and fig. 10, and the particle size of the prepared nano-selenium particles is between 100 and 200 nm.
Example 1: sensitivity of Bacillus amyloliquefaciens Lxz-41 to utilization of different C sources and chemical factors
Inoculating bacillus amyloliquefaciens Lxz-41 into LB solid culture medium, culturing at 28 ℃ for 16h, scraping 2-ring colonies, inoculating into LB liquid culture medium, and culturing at 28 ℃ for 24 h.
The culture broth was added to 96-well plates with different C sources and sensitive factors using a shotgun, and the layout of the 96-well plates is shown in table 1 below.
TABLE 1
Standing at 28 deg.C for 48h, and reading the light absorption value of color reaction with a microorganism biochemical analyzer, as shown in figure 6.
Example 2: identification of Bacillus amyloliquefaciens Lxz-41
Inoculating bacillus amyloliquefaciens Lxz-41 into LB solid culture medium, culturing at 28 ℃ for 16h, scraping 2-ring colonies, inoculating into LB liquid culture medium, and culturing at 28 ℃ for 24 h.
Adding 1ml of bacillus amyloliquefaciens Lxz-41 culture solution into a sterilized EP tube, centrifuging at 12000r/min for 1min, and extracting the DNA of the bacillus amyloliquefaciens Lxz-41 according to a bacterial extraction kit of Tiangen Biochemical technology limited company.
And (3) PCR reaction conditions: primer: 27F: AGTTTGATCMTGGCTCAG, 1492R: GGTTACCTTGTTACGACTT reaction conditions are shown in Table 2
TABLE 2
And (3) carrying out kit purification on the PCR product, then sequencing, carrying out fragment splicing by using DNAMMAN 5.0 after sequencing, comparing the obtained sequence with the attached drawing and 16S rDNA BLAST on GENbank, and displaying that the sequence similarity of the strain Lxz-41 and Bacillus amyloliquefaciens Y2 reaches 100 percent according to the result. Therefore, Lxz-41 is determined to be Bacillus amyloliquefaciens named as Bacillus amyloliquefaciens Lxz-41 and is preserved in China center for type culture Collection with the preservation number of CCTCC M2016578.
Example 3: tolerance concentration of bacillus amyloliquefaciens Lxz-41 to sodium selenite
Inoculating bacillus amyloliquefaciens Lxz-41 into LB solid culture medium, culturing at 28 ℃ for 16h, scraping 2-ring colonies, inoculating into LB liquid culture medium, and culturing at 28 ℃ for 24 h.
Preparing beef extract peptone or LB liquid culture medium containing sodium selenite with concentration of 0, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000mg/L respectively. Taking sterilized 96-well plates, enabling each concentration to be 8 in parallel, adding 150uL of culture medium into each well, inoculating 5uL of bacillus amyloliquefaciens Lxz-41, and carrying out shake culture at 30 ℃ and 100r/min for 48 h. The results in FIG. 8 show that the thalli grow at sodium selenite concentrations of 4500mg/L and below, the sodium selenite content in the solution begins to turn red at a concentration of above 100mg/L, and the growth of the thalli is already obviously inhibited at sodium selenite concentrations of 4500mg/L and above. Therefore, the maximum tolerance concentration of Bacillus amyloliquefaciens Lxz-41 to sodium selenite was 4500 mg/L.
Example 4: preparation of nano-selenium
Activating strains: the preserved strain is inoculated on a beef extract peptone test tube agar slant culture medium, static culture and activation are carried out at 25 ℃, and the slant is full of bacterial colonies.
Seed culture: inoculating slant culture strain into beef extract peptone liquid culture medium containing 100mg/L sodium selenite, and shake culturing at 28 deg.C and 80r/min for 24 hr to obtain seed liquid, wherein the liquid content is 20% of the container capacity.
Controllable production of nano-selenium: inoculating the seed culture solution into a 1L triangular flask fermentation culture medium according to the inoculation amount of 5%, wherein the liquid loading amount is 400mL, adding 500mg/L sodium selenite before the fermentation starts, adding 250mg/L sodium selenite after 24h, and performing shake-flask culture at 25 ℃ for 48h at 80 r/min.
Extracting and purifying nano selenium: freezing and centrifuging fermentation liquor 10000r/min for 10min, collecting thalli, washing the thalli with 100mL sterile physiological saline for three times, suspending the thalli with 20mL sterile distilled water, performing ice bath ultrasonic wall breaking, performing 30W, starting and stopping the operation at an interval of 15s, crushing the thalli for 20min, freezing and centrifuging thalli lysate 10000r/min for 10min, cleaning precipitates with 20mL deionized water for three times, suspending the precipitates with 10mL sterile distilled water to obtain nano-selenium suspension, adding chloroform with the same volume for extraction for 30min, extracting for 3 times, combining lower-layer water phases, homogenizing the precipitates with a homogenizer for 5min, freezing and centrifuging at 10000r/min for 10min, cleaning the precipitates with 10mL sterile physiological saline for 3 times, and freeze-drying for 24h to obtain the microbial nano-selenium.
And (3) detecting the purity and the average particle size of the nano selenium: the detection of the selenium is carried out according to the national standard GB 5009.93-2017, under the condition, the conversion rate of the nano selenium is 88.31%, the purity of the obtained selenium is 91.2%, and the average particle size is 153 +/-20 nm.
Example 5: preparation of nano-selenium
Activating strains: the preserved strain is inoculated on a beef extract peptone test tube agar slant culture medium, and static culture and activation are carried out at 40 ℃ until the slant is full of bacterial colonies.
Seed culture: inoculating slant culture strain into beef extract peptone liquid culture medium containing 100mg/L sodium selenite, and shake culturing at 40 deg.C and 200r/min for 24 hr to obtain seed liquid, wherein the liquid content is 20% of the container capacity.
Controllable production of nano-selenium: inoculating the seed culture solution into a 500L fermentation medium of a fermentation tank according to the inoculation amount of 5%, wherein the liquid loading amount is 350L, and the continuous feeding of sodium selenite mother liquor (50g/L) is adopted, the feeding speed is 1.5mL/min, the continuous feeding is 36h, and the fermentation culture is carried out for 72h at 40 ℃ and 200 r/min.
Extracting and purifying nano selenium: freezing and centrifuging 4000r/min fermentation broth of a fermentation tank for 20min, collecting thalli, washing three times by using 100L of sterile physiological saline, suspending by using 15L of sterile distilled water, performing ice bath ultrasonic wall breaking, 800W, starting and stopping at an interval of 15s, crushing for 20min, freezing and centrifuging 5000r/min thalli lysate for 20min, cleaning precipitates by using 15L of deionized water for three times, suspending by using 5L of distilled water to obtain nano-selenium suspension, adding chloroform with the same volume for extraction for 30min, extracting for 3 times, combining lower-layer aqueous phases, homogenizing by using a homogenizer for 5min, freezing and centrifuging for 10min at 5000r/min, cleaning the precipitates by using 5L of sterile physiological saline for 3 times, and freeze-drying for 48h to obtain the microbial nano-selenium.
And (3) detecting the purity and the average particle size of the nano selenium: the detection of selenium is carried out according to the national standard GB 5009.93-2017; under the condition, the conversion rate of the nano selenium is 94.32 percent, the purity of the obtained selenium is 91.6 percent, and the average grain diameter is 116 +/-20 nm.
Example 6: preparation of nano-selenium
Activating strains: the preserved strain is inoculated on a beef extract peptone test tube agar slant culture medium, static culture and activation are carried out at 30 ℃, and the slant is full of bacterial colonies.
Seed culture: inoculating slant culture strain into beef extract peptone liquid culture medium containing 100mg/L sodium selenite, loading liquid amount of 20% of container capacity, and performing shake culture at 30 deg.C and 140r/min for 24 hr to obtain seed liquid.
Controllable production of nano-selenium: inoculating the seed culture solution into a fermentation culture medium of a 100L small fermentation tank according to the inoculation amount of 5%, wherein the liquid loading amount is 50L, adding 500mg/L sodium selenite before the fermentation starts, continuously adding sodium selenite mother liquor (50g/L) after 24h at 0.1mL/min, continuously adding 24h, and stirring at 30 ℃ for 140r/min for co-culture for 60 h.
Extracting and purifying nano selenium: freezing and centrifuging fermentation liquor at 8000r/min for 15min, collecting thallus, washing with 10L sterile physiological saline for three times, suspending with 2L sterile distilled water, performing ice bath ultrasonic wall breaking, 600W, starting and stopping at an interval of 15s, crushing for 20min, freezing and centrifuging thallus lysate at 8000r/min for 15min, cleaning precipitate with 1L deionized water for three times, suspending with 1L distilled water to obtain nano-selenium suspension, adding chloroform with the same volume for extraction for 30min, extracting for 3 times, combining lower-layer water phases, homogenizing with a homogenizer for 5min, freezing and centrifuging at 8000r/min for 15min, cleaning precipitate with 1L sterile physiological saline for 3 times, and freeze-drying for 48h to obtain the microbial nano-selenium.
And (3) detecting the purity and the average particle size of the nano selenium: the detection of selenium is carried out according to the national standard GB 5009.93-2017, under the condition, the conversion rate of nano selenium is 91.3%, the purity of the obtained selenium is 92%, and the average particle size is 145 +/-20 nm.
Example 7: preparation of nano-selenium
Activating strains: the preserved strain is inoculated on a beef extract peptone test tube agar slant culture medium, static culture and activation are carried out at 35 ℃, and the slant is full of bacterial colonies.
Seed culture: inoculating slant culture strain into beef extract peptone liquid culture medium containing 100mg/L sodium selenite, and shake culturing at 35 deg.C and 180r/min for 24 hr to obtain seed liquid, wherein the liquid content is 20% of the container capacity.
Controllable production of nano-selenium: inoculating the seed culture solution into a fermentation medium of a 1T fermentation tank according to the inoculation amount of 5%, wherein the liquid loading amount is 600L, the flow rate is 2mL/min in a manner of continuously feeding sodium selenite mother solution (50g/L), the flow rate is 1mL/min after continuously feeding for 48h, the flow rate is 12h, and the co-fermentation culture is carried out for 72h at 35 ℃ and 180 r/min.
Extracting and purifying nano selenium: freezing and centrifuging the fermentation liquor for 20min at 5000r/min, collecting thalli, washing the thalli with 150L of sterile physiological saline for three times, suspending the thalli with 30L of sterile distilled water, performing ice bath ultrasonic wall breaking, performing 1000W, starting and stopping intervals of 15s, crushing for 20min, freezing and centrifuging thalli lysate for 20min at 5000r/min, cleaning precipitates with 15L of deionized water for three times, suspending the precipitates with 15L of distilled water to obtain nano-selenium suspension, adding chloroform with the same volume for extraction for 30min, extracting for 3 times, combining lower-layer water phases, homogenizing for 5min with a homogenizer, freezing and centrifuging for 20min at 5000r/min, cleaning the precipitates with 15L of sterile physiological saline for 3 times, and freezing and drying for 48h to obtain the microbial nano-selenium.
And (3) detecting the purity and the average particle size of the nano selenium: the detection of selenium is carried out according to the national standard GB 5009.93-2017, under the condition, the conversion rate of nano selenium is 90.8%, the purity of the obtained selenium is 93.2%, and the average particle size is 153 +/-20 nm.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
SEQUENCE LISTING
<110> institute for microorganisms of Shaanxi province
<120> bacillus amyloliquefaciens Lxz-41 and method for preparing nano-selenium controllably by using same
<130> 2017
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1404
<212> DNA
<213> Bacillus amyloliquefaciens Lxz-41 (Bacillus amyloliquefaciens Lxz-41)
<400> 1
aatacatgca agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg 60
tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa 120
taccggatgc ttgtttgaac cgcatggttc agacataaaa ggtggcttcg gctaccactt 180
acagatggac ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcaacgat 240
gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct 300
acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc 360
gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtgccgtt 420
caaatagggc ggcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc 480
agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc 540
aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa 600
ctggggaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta 660
gagatgtgga ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga 720
gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780
gtgctaagtg ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg 840
cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg 900
gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc 960
tgacaatcct agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg 1020
tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct 1080
tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg 1140
tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg 1200
acagaacaaa gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt 1260
cggatcgcag tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc 1320
atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt 1380
gtaacacccg aagtcggtga ggta 1404
Claims (10)
1. A Bacillus amyloliquefaciens Lxz-41 with a preservation number of: CCTCC NO: m2016578, deposited in the China center for type culture Collection, that has the ability to produce nano-selenium in a controlled manner.
2. A method for controllable production of nano-selenium by using Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Lxz-41 as claimed in claim 1, which is characterized by comprising the following steps:
s1, activating strains: inoculating the preserved strain to a beef extract peptone or LB test tube agar slant culture medium, and performing static culture and activation at 25-40 ℃ until the slant surface is full of bacterial colonies;
s2, seed culture: inoculating slant culture strain into beef extract peptone liquid culture medium containing selenium salt, loading liquid amount is 20% of container capacity, and performing shake culture at 25-40 deg.C and 80-200r/min for 12-24 hr to obtain seed liquid;
s3, controllable production of nano-selenium: inoculating the seed culture solution into a fermentation container filled with a fermentation culture medium according to the inoculation amount of 1-5%, wherein the fermentation culture medium comprises selenium salt, the selenium salt is added in a fed-batch manner, the liquid loading amount of the fermentation culture medium is 40-70% of the capacity of the fermentation container, the stirring speed is 50-200r/min, the ventilation volume is 0.5-2.0vvm, the temperature is kept at 25-40 ℃, and the fermentation time is 24-72 h;
s4, extracting and purifying nano selenium: and (2) putting the fermentation liquid in a tank, performing refrigerated centrifugation at the temperature of 4000 plus 10000r/min4 ℃ for 10-20min, collecting the obtained thallus, washing the thallus with sterile normal saline with the volume of about 1/4 fermentation liquid for three times, suspending the thallus with sterile distilled water with the volume of about 1/20, performing ice bath ultrasonic wall breaking for 20-40min, performing refrigerated centrifugation at the temperature of 4 ℃ for 10-20min on thallus lysate 4000 plus 10000r/min, cleaning the precipitate with deionized water with the same volume for three times, suspending the precipitate with distilled water with the volume of about 1/2 to obtain nano-selenium suspension, adding chloroform with the same volume for extraction for 30min, extracting for 2-3 times, combining lower-layer water phases, homogenizing the precipitate with a homogenizer for 2-5min, performing refrigerated centrifugation at the temperature of 4000 plus 10000r/min4 ℃ for 10-20min, cleaning the precipitate with equal-volume sterile normal saline for 3 times, and performing refrigerated drying to obtain the microbial nano-selenium.
3. The method as claimed in claim 2, wherein the selenium salt in the liquid culture medium of beef extract peptone in S2 is one or more of selenite and selenate, and the mass-to-volume ratio is 500mg/L of 100-.
4. The method as claimed in claim 2, wherein the selenium salt in the culture medium of S2 or S3 is separately sterilized and then added to the sterilized culture medium.
5. The method of claim 2, wherein the fermentation medium in S3 has a composition (g/L): glucose 10, peptone 5, ammonium sulfate 1, magnesium sulfate heptahydrate 0.3, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate 0.5, sodium chloride 2, selenium salt 0.1-4.5, surfactant 0.5-1, and pH = 7.2-7.4.
6. The method as claimed in claim 5, wherein the selenium salt in the fermentation medium in S3 is fed batch or continuous fed batch with mother liquor to control the concentration of selenium salt in the fermentation broth, and thus the transformation efficiency of the strain, wherein the selenium salt is one or more of selenite and selenate.
7. The method as claimed in claim 6, wherein sodium selenite is fed in batch or continuous manner to the fermentation medium, and the concentration of the mother liquor of sodium selenite is 50-500 g/L.
8. The method of claim 2, wherein in S4, the power of ultrasonic crushing is 400W-1000W, the frequency is 20-40KHz, the start-stop interval is 10-15S, and the crushing is 20-40 min.
9. The method according to claim 5, wherein the surfactant is one or more of SDS, Tween 40, Tween 60 and Tween 80.
10. A complex microbial inoculant comprising Bacillus amyloliquefaciens Lxz-41 according to claim 1.
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| CN111235066B (en) * | 2020-03-12 | 2022-04-22 | 陕西省微生物研究所 | Bacillus belgii Lxh and method for preparing agricultural nano-selenium tablet by using same |
| CN113373180B (en) * | 2020-12-18 | 2023-05-26 | 中国农业科学院烟草研究所 | Nano-selenium synthesized active bacterial liquid of bacillus amyloliquefaciens, and preparation method and application thereof |
| CN114946884A (en) * | 2022-06-30 | 2022-08-30 | 陕西科技大学 | Nano-selenium probiotic preparation and preparation method and application thereof |
| CN115851544B (en) * | 2022-12-16 | 2024-02-09 | 陕西省微生物研究所 | Bacillus subtilis and method for preparing selenium-enriched natto by using same |
| CN115927114B (en) * | 2022-12-27 | 2023-10-13 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Selenite reducing bacteria, method for synthesizing nano-selenium by using selenite reducing bacteria and application of selenite reducing bacteria |
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