CN107881127A - A kind of bacillus amyloliquefaciens Lxz 41 and the method using the bacterial strain controllable preparation nanometer selenium - Google Patents
A kind of bacillus amyloliquefaciens Lxz 41 and the method using the bacterial strain controllable preparation nanometer selenium Download PDFInfo
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Abstract
A kind of method the invention provides bacillus amyloliquefaciens Lxz 41 and using the bacterial strain controllable preparation nanometer selenium.Bacillus amyloliquefaciens Lxz 41 deposit number is：CCTCC M2016578, are deposited in China typical culture collection center, and the bacterial strain has the ability of controllable production nanometer selenium.The present invention is using bacillus amyloliquefaciens Lxz 41 as fermentation strain, stream plus selenate, and control strain culturing time, rotating speed, surfactant concentration carry out controllable preparation nanometer selenium, collects fermentation liquid precipitate, obtains nano granules of selenium after purification.The selenite of bacterial strain resisting high-concentration of the present invention, caused most nano granules of selenium particle diameter can be made in below 200nm by the control of preparation process condition.The controllable nano selenium preparation method of the present invention can mitigate inhibitory action of the selenate to thalli growth, increase the conversion ratio of nanometer selenium, the effectively clustering phenomena of control nano granules of selenium, and required culture medium and condition of culture are simple, cost is low, easy to operate are suitable for the production of large-scale fermentation tank.
Description
Technical field
The invention belongs to microorganism application technology and nanometer selenium preparation field, a kind of bacillus amyloliquefaciens are specifically related to
Lxz-41 and the method using the bacterial strain controllable preparation nanometer selenium.
Background technology
Selenium is very important nutrient in the ecosystem, there is the title of " biological element ".Early in nineteen fifty-seven Schwarz
Determine nutrient necessary to selenium is humans and animals.1973, Rotruek et al. had found that selenium is higher mammal biological metabolism
The active component of indispensable glutathione peroxidase.The same year, selenium assert it is needed by human by the World Health Organization
Trace element.Hereafter substantial amounts of research shows that the nutrition scope of selenium is very narrow.Human body Selenium In Foods content is less than 0.05mg/
Kg will result in selenium deficiency, and poisoning can be produced again more than 5mg/kg.Selenium skewness on earth, there is the high selenium area in part
With serious selenium deficiency area.The area of China 72% deficiency of selenium intake in selenium deficiency, low selenium band, meals drastically influence several hundred million
The health of population.According to Chinese Soclety of Nutrition's survey report, daily selenium intake of being grown up is only 26.63ug, away from China
Nutrition Society and international selenium association recommend daily intaking amount 50ug to differ greatly.Selenium deficiency can cause the Keshan disease of the mankind, Kaschin-Beck disease,
The diseases such as cancer, diabetes, cardiovascular and cerebrovascular diseases, sterility, and appropriate selenium-supply can give protection against cancer, be antitumor, AIDS resisting and anti-
Aging.Selenium deficiency the health of people and cause potential hazard by serious threat.In recent years, as people increasingly focus on protecting
Strong and health, various selenium-rich products arise at the historic moment, but product quality is uneven, the unified mark of the content neither one of selenium
It is accurate.And due to the inequality of Se content in soil, selenium-rich product is in planting process all using sprinkling inorganic selenium (sodium selenate, Asia
Sodium selenate) improve the content of selenium in product.But inorganic selenium (sodium selenate, sodium selenite) toxicity is big, the play of the 6th class is listed in
Noxious material, the use of inorganic selenium have seriously endangered environment and human health.Therefore, primary Task is that exploitation is a kind of high now
The selenium source of performance hypotoxicity, this by be selemium nutrition health care study emphasis
Begin one's study and prepare the selenium-containing compound of high activity hypotoxicity, the progress of this respect is slower at present.Pass
Include the selenium of two kinds of forms in meaning of uniting：First, the selenium compound of activity and toxicity is had concurrently, second, the zeroth order selenium that toxicity is low.Wherein
Zeroth order selenium is primarily present three kinds of grey, red and black forms, for the elemental selenium of these three forms, known to us
Grey and black selenium particle diameter are big, and do not have bioactivity, and Nano red selenium has bioactivity, according to acute toxicity
(LD50) data show, inorganic selenium LD50=15mg/kg, Organic Selenium LD50=30-40mg/kg, nanometer selenium LD50=113mg/kg.
So far, nanometer selenium has found that toxicity is minimum.The production method of nanometer selenium mainly has chemical synthesis and biosynthesis at present
Method, biological nanometer selenium than chemical synthesis nanometer selenium evenly, form is more regular, and the nanometer selenium high temperature resistant of biosynthesis,
It is more stable, it is not easy to change into black or brown nanometer selenium.Biosynthesis nanometer selenium is mainly produced using micro-reduction selenite
Raw, with deepening continuously for research, it is extremely abundant to synthesize the kind that the microorganism of nanometer selenium is covered, more than 30 category, bag
Include Escherichia coli (Escherichia coli), Rhodobacter capsulatus (Rhodobacter Imhoff), bacillus subtilis
(Bacillus subtilis) etc..But because most of microbe is not easy fermenting and producing, characteristic not easy to maintain, it is impossible to real
Existing industrialized production.The bacillus gemma stronger due to resistance can be produced, the bad ring being resistant in process of manufacture
Border and condition, it is the good microorganism fungus kind of industrialized production nanometer selenium plus being easy to preserve.But due to various factors, lead
Microorganism nano granules of selenium particle diameter is larger and not of uniform size caused by cause.
The content of the invention
It is an object of the invention to provide a kind of bacillus amyloliquefaciens Lxz-41, the bacillus amyloliquefaciens have controllable
The ability of standby nanometer selenium.
The technical solution adopted by the present invention is：
A kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Lxz-41, deposit number are：CCTCC
M2016578, China typical culture collection center is deposited in, the bacterial strain has the ability of controllable production nanometer selenium.
A kind of controllable production nanometer seleniums of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Lxz-41
Method, comprise the steps of:
S1. actication of culture：Preservation strain is connected on beef extract-peptone or LB test tube agar slant culture-mediums, 25-40
The activation of DEG C static gas wave refrigerator, treats that bacterium colony covers with inclined-plane；
S2. seed culture：Inclined-plane culture strain is inoculated in the beef extract-peptone fluid nutrient medium containing selenium salt, filled
Liquid measure is 20%, 25-40 DEG C, 80-200r/min shaken cultivation 12-24h of container capacity, obtains seed liquor；
S3. the controllable production of nanometer selenium：Seed culture fluid is seeded to equipped with fermentation medium according to 1%-5% inoculum concentrations
In round, the fermentation medium includes selenium salt, and selenium salt is added using stream plus form, and fermentation medium liquid amount is hair
The 40%-70% of ferment container, stir speed (S.S.) 50-200r/min, throughput 0.5-2.0vvm, temperature are kept for 25-40 DEG C, during fermentation
Between 24-72h；
S4. nanometer selenium extraction purification：Tank under zymotic fluid, 4000-10000r/min4 DEG C of refrigerated centrifuge 10-20min, collect
Thalline is obtained, is flushed three times with about 1/4 fermentating liquid volume sterile saline, is hanged with about 1/20 volume sterile distilled water
It is floating, carry out ice-bath ultrasonic broken wall 20-40min, 4000-10000r/min4 DEG C of refrigerated centrifuge 10-20min of cellular lysate liquid, precipitation
Cleaned three times with same volume deionized water, and aqueous suspension is distilled with about 1/2 volume, obtain nanometer selenium suspension, add same volume chlorine
It is imitative to carry out extraction 30min, extract 2-3 time, merging lower floor aqueous phase, with homogenizer homogeneous 2-5min, 4000-10000r/min4 DEG C
Refrigerated centrifuge 10-20min, precipitation are cleaned 3 times with isometric sterile saline, are freeze-dried and produce microorganism nanometer selenium.
Further, the selenium salt in S2 in beef extract-peptone fluid nutrient medium is one kind in selenite, selenate
Or several, mass volume ratio 100-500mg/L.
Selenium salt in S2, S3 culture medium is individually sterilized, is then added in the culture medium after sterilizing.
Further, fermentation medium component is (g/L) in S3：Glucose 10, peptone 5, ammonium sulfate 1, seven water sulfuric acid
Magnesium 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, pH=
7.2-7.4。
Further, the selenium salt in S3 in fermentation medium is flowed in batches with mother liquor adds form or continuous stream to add form, with control
The concentration of selenium salt in zymotic fluid processed, and then the transformation efficiency of bacterial strain is controlled, the selenium salt is one kind in selenite, selenate
It is or several.
Further, in fermentation medium in batches stream plus or continuous stream add sodium selenite, sodium selenite mother liquor used it is dense
Spend for 50-500g/L.
Further, ultrasonication power 400W-1000W in S4, frequency 20-40KHz, start and stop interval 10-15s, crush
20-40min。
Further, the surfactant is the one or more in SDS, polysorbate40, polysorbate60, Tween 80.
The present invention possesses advantages below：
1. bacillus amyloliquefaciens Lxz-41 used in the present invention, 5000mg/L sodium selenite is resistant to, higher than before
The tolerable concentration of the bacillus of report, and the present invention is using stream plus selenite form in batches, according to the growth rhythm of thalline,
The selenite of low concentration is added in laundering period early stage, with the growth of thalline, the concentration of selenite gradually rises.Such energy
Enough solve the problems, such as the disposable addition due to selenite to being poisoned produced by thalline, and can make under conditions of stream adds
The concentration of selenite maintains under the optimal conversion concentration of thalline always, improves the transformation efficiency of nanometer selenium.
2. the present invention is by controlling stir speed (S.S.) in fermentation process, surfactant concentration, fermentation time to control
Obtain the size distribution of nanometer selenium particle diameter.And stir speed (S.S.) is bigger, particle diameter is smaller；Do not influenceing in thalli growth concentration range, table
Face surfactant concentration is bigger, and particle diameter is smaller；Do not influenceing in the range of thalli growth, fermentation time is shorter, and particle diameter is smaller.
3. bacillus amyloliquefaciens Lxz-41 selected by the present invention can resisting high-concentration sodium selenite, its strain belongs to benefit
Raw bacterium is safe to use, and bacillus amyloliquefaciens metabolite enriches, and the antibiotic antibacterial protein of secretion or polypeptides matter etc. add
It is added in fertilizer, preferable biological control and selenium-rich effect can be played；Also livestock and poultry feeding can be added to directly as feed addictive
In material.Bacillus amyloliquefaciens Lxz-41 condition of culture is extensive, is not easy microbiological contamination, and cost is low, and nanometer selenium is prepared using bacterial strain is changed
Method has the advantages that simple to operate, cost is low, prepares that convenient, yield is high, is adapted to large-scale prepare.
Brief description of the drawings
When Fig. 1 is for stream of the invention plus with disposable addition sodium selenite, selenite concentration and pair of nanometer selenium conversion ratio
Compare block diagram；
When Fig. 2 is for stream of the invention plus with disposable addition sodium selenite, pair of selenite concentration and thalli growth situation
Compare block diagram；
Fig. 3 is the contrast block diagram for the average grain diameter change that nanometer selenium is prepared under the different stir speed (S.S.)s of the present invention；
Fig. 4 is the contrast block diagram for the average grain diameter change that nanometer selenium is prepared under the different fermentations time of the present invention；
Fig. 5 is the contrast block diagram for the average grain diameter change that nanometer selenium is prepared under different surfaces surfactant concentration of the present invention；
Fig. 6 is strain characteristic figure of the present invention；
Fig. 7 is bacterial strain of the present invention to sodium selenite tolerable concentration experimental result；
Fig. 8 is that strain liquid shake flask fermentation of the present invention produces nanometer selenium photo；
Fig. 9 is electron microscope photo scanning of the nanometer selenium in the case where resolution ratio is 1 μm prepared by the inventive method；
Figure 10 is electron microscope photo scanning of the nanometer selenium in the case where resolution ratio is 100nm prepared by the inventive method.
Embodiment
The present invention is described in further detail below by embodiment combination accompanying drawing.Those skilled in the art can
With recognizing without lifting an eyebrow, which part feature is dispensed in varied situations, or can be by other elements, material
Material, method are substituted.In some cases, the related certain operations of the application do not show or described in the description,
This is to be flooded in order to avoid the core of the application by excessive description, and to those skilled in the art, in detail
It is not necessary to describe these associative operations, and they are at the general technology knowledge of description and this area in specification
Associative operation can completely be understood.
In addition, feature described in this description, operation or feature can combine to form respectively in any suitable way
Kind embodiment.Meanwhile each step in method description or action can also can be aobvious and easy according to those skilled in the art institute
The mode carry out order exchange or adjustment seen.Therefore, the various orders in specification and drawings are intended merely to clearly describe a certain
Individual embodiment, necessary order is not meant to be, wherein some sequentially must comply with unless otherwise indicated.
It should be noted that the detection method of average grain diameter is as follows in present specification：
Choose three groups parallel (nano granules of selenium obtained under three groups of identical preparation conditions) to be shot, in every photo
Randomly choose and measure Ni nanometer selenium particle diameter, average grain diameter is calculated by formula below.
The solution of the present invention one plant of tolerable higher concentration sodium selenite isolated from the soil of Shaanxi Province Ankang City Ziyang
Bacillus amyloliquefacienses (Bacillus amyloliquefaciens) Lxz-41, bacterial strain Lxz-41 is now preserved in Chinese Typical Representative culture
Thing collection, address：Wuchang, wuhan road Ka mountain, postcode 430072, deposit number CCTCC M2016578, preservation date
On October 18th, 2016.
The following institute of 16srDNA sequences of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Lxz-41
Show：
AATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACA
CGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATG
GTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGG
CTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCT
ACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTT
TCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAG
AAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAA
AGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAA
CTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCG
AAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGT
CCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCC
GCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT
AATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGG
GGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAA
CCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT
GACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCG
CGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCG
CTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGT
TTGTAACACCCGAAGTCGGTGAGGTA
The present invention also provides one kind and utilizes bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Lxz-41
The method of controllable preparation nanometer selenium, this method comprise the following steps：
S1. actication of culture：Preservation strain is connected on beef extract-peptone or LB test tube agar slant culture-mediums, 25-40
The activation of DEG C static gas wave refrigerator, treats that bacterium colony covers with inclined-plane；
S2. seed culture：Inclined-plane culture strain is inoculated in the beef extract-peptone fluid nutrient medium containing selenium salt, filled
Liquid measure is 20%, 25-40 DEG C, 80-200r/min shaken cultivation 12-24h of container capacity, obtains seed liquor；
S3. the controllable production of nanometer selenium：Seed culture fluid is seeded to equipped with fermentation medium according to 1%-5% inoculum concentrations
In round, the fermentation medium includes selenium salt, and selenium salt is added using stream plus form, and fermentation medium liquid amount is hair
The 40%-70% of ferment container, stir speed (S.S.) 50-200r/min, throughput 0.5-2.0vvm, temperature are kept for 25-40 DEG C, during fermentation
Between 24-72h；
S4. nanometer selenium extraction purification：Tank under zymotic fluid, 4000-10000r/min4 DEG C of refrigerated centrifuge 10-20min, collect
Thalline is obtained, is flushed three times with about 1/4 fermentating liquid volume sterile saline, is hanged with about 1/20 volume sterile distilled water
It is floating, carry out ice-bath ultrasonic broken wall 20-40min, 4000-10000r/min4 DEG C of refrigerated centrifuge 10-20min of cellular lysate liquid, precipitation
Cleaned three times with same volume deionized water, and aqueous suspension is distilled with about 1/2 volume, obtain nanometer selenium suspension, add same volume chlorine
It is imitative to carry out extraction 30min, extract 2-3 time, merging lower floor aqueous phase, with homogenizer homogeneous 2-5min, 4000-10000r/min4 DEG C
Refrigerated centrifuge 10-20min, precipitation are cleaned 3 times with isometric sterile saline, are freeze-dried and produce microorganism nanometer selenium.
Selenium salt in S2 in beef extract-peptone fluid nutrient medium is selenite, the one or more in selenate, matter
Amount volume ratio is 100-500mg/L.
Selenium salt in S2, S3 culture medium is individually sterilized, is then added in the culture medium after sterilizing.
Fermentation medium component is (g/L) in S3：Glucose 10, peptone 5, ammonium sulfate 1, epsom salt 0.3, phosphoric acid
Potassium dihydrogen 0.5, dipotassium hydrogen phosphate 0.5, sodium chloride 2, selenium salt 0.1-4.5, surfactant 0.5-1, pH=7.2-7.4.
Selenium salt in S3 in fermentation medium is flowed in batches with mother liquor adds form or continuous stream to add form, to control in zymotic fluid
The concentration of selenium salt, and then the transformation efficiency of bacterial strain is controlled, the selenium salt is the one or more in selenite, selenate.
Stream adds in batches in fermentation medium or continuous stream adds sodium selenite, and the concentration of sodium selenite mother liquor used is 50-
500g/L。
Ultrasonication power 400W-1000W in S4, frequency 20-40KHz, start and stop interval 10-15s, crush 20-40min.
The surfactant is the one or more in SDS, polysorbate40, polysorbate60, Tween 80.
Fig. 7 be bacterial strain of the present invention to sodium selenite tolerable concentration experimental result, from figure, bacillus amyloliquefaciens energy
5000mg/L sodium selenite is enough resistant to, and the concentration range for being best suitable for the sodium selenite of thalli growth is 1500mg/L-
3000mg/L, the tolerance of the high sodium selenite of bacterial strain improve the production capacity of nanometer selenium.
Referring to Fig. 1 and Fig. 2, by taking the 48h that ferments as an example, when stream adds and disposably adds seed culture fluid, selenite is dense
The contrast block diagram of degree and nanometer selenium conversion ratio；From in figure, adding selenite form using stream in batches, according to the life of thalline
Long rule, the selenite of low concentration is added in laundering period early stage, with the growth of thalline, the concentration of selenite gradually rises
It is high.So can solve the problem that the problem of disposable addition due to selenite produced by thalline to poisoning, and stream plus bar
Under part the concentration of selenite can maintained under the optimal conversion concentration of thalline always, improves the transformation efficiency of nanometer selenium.
Referring to Fig. 3-Fig. 5, by controlling stir speed (S.S.) in fermentation process, surfactant concentration, fermentation time to control
The size distribution of system gained nanometer selenium particle diameter.And stir speed (S.S.) is bigger, particle diameter is smaller；Do not influenceing thalli growth concentration range
Interior, surfactant concentration is bigger, and particle diameter is smaller；Do not influenceing in the range of thalli growth, fermentation time is shorter, and particle diameter is smaller.
Shaken eventually through bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) Lxz-41 liquid
Bottle fermentation produces nanometer selenium photo as shown in figure 8, using sodium selenite to add concentration as 2000mg/L, 3000mg/L, 140r/min,
28 DEG C of culture 24h, solution become cerise；By collecting the liquid precipitate that ferments, nano granules of selenium microstructure ginseng is obtained after purification
As shown in Fig. 9 and Figure 10, prepared nano granules of selenium particle diameter is between 100-200nm.
Embodiment 1：The sensitiveness of utilizations and chemokines of the bacillus amyloliquefaciens Lxz-41 to different C sources
Bacillus amyloliquefaciens Lxz-41 is inoculated in LB solid mediums, 28 DEG C of culture 16h, 2 ring bacterium colonies of scraping connect
Kind is in LB fluid nutrient mediums, 28 DEG C of culture 24h.
Nutrient solution is added in 96 orifice plates with different C sources and sensitive factor with the volley of rifle fire, 96 orifice plate layouts such as following table
1。
Table 1
28 DEG C of constant temperature static gas wave refrigerator 48h, chromogenic reaction light absorption value is read by microbial biochemical analyzer, sees accompanying drawing 6.
Embodiment 2：Bacillus amyloliquefaciens Lxz-41 identification
Bacillus amyloliquefaciens Lxz-41 is inoculated in LB solid mediums, 28 DEG C of culture 16h, 2 ring bacterium colonies of scraping connect
Kind is in LB fluid nutrient mediums, 28 DEG C of culture 24h.
Take the EP of sterilizing to manage, add bacillus amyloliquefaciens Lxz-41 nutrient solutions 1ml, 12000r/min centrifugation 1min, so
Bacillus amyloliquefaciens Lxz-41DNA extraction is carried out according to Tiangeng biochemical technology Co., Ltd bacterium extracts kit afterwards.
PCR reaction conditions：Primer：27F:AGTTTGATCMTGGCTCAG, 1492R:GGTTACCTTGTTACGACTT. it is anti-
Condition is answered to be shown in Table 2
Table 2
PCR primer is sequenced after carrying out kits, is carried out fragment assembly using DNAMAN5.0 after sequencing, will be obtained
Sequence, see accompanying drawing and carry out 16S rDNA BLAST in GENbank comparing, shown according to result, bacterial strain Lxz-41 with
Bacillus amyloliquefaciens Y2, sequence similarity reach 100%.Thereby determine that Lxz-41 for solution starch bud
Spore bacillus, name as Bacillus amyloliquefaciens Lxz-41, and be preserved in China typical culture collection
The heart, preserving number CCTCC M 2016578.
Embodiment 3：Tolerable concentrations of the bacillus amyloliquefaciens Lxz-41 to sodium selenite
Bacillus amyloliquefaciens Lxz-41 is inoculated in LB solid mediums, 28 DEG C of culture 16h, 2 ring bacterium colonies of scraping connect
Kind is in LB fluid nutrient mediums, 28 DEG C of culture 24h.
Configuration containing concentration of sodium selenite is respectively 0,100,500,1000,1500,2000,2500,3000,3500,4000,
4500th, 5000mg/L beef extract-peptone or LB fluid nutrient mediums.96 orifice plates of sterilizing are taken, each concentration 8 is parallel, per hole
Add 150uL culture mediums, access 5uL bacillus amyloliquefaciens Lxz-41,30 DEG C, 100r/min shaken cultivations 48h.The knot of accompanying drawing 8
Fruit shows, concentration of sodium selenite 4500mg/L and following, and thalline has grown, and solution sodium selenite content is in 100mg/L
Start to redden above, concentration of sodium selenite substantially suppresses the growth of thalline in 4500mg/L and the above.Accordingly, it is determined that
Bacillus amyloliquefaciens Lxz-41 to the maximal tolerable concentration of sodium selenite is 4500mg/L.
Embodiment 4：It is prepared by nanometer selenium
Actication of culture：Preservation strain is connected on beef extract-peptone test tube agar slant culture-medium, 25 DEG C of static gas wave refrigerators
Activation, treats that bacterium colony covers with inclined-plane.
Seed culture：Inclined-plane culture strain is inoculated in into the beef extract-peptone liquid containing 100mg/L sodium selenites to train
Support in base, liquid amount is the 20% of container capacity, 28 DEG C, 80r/min shaken cultivation 24h, obtains seed liquor.
The controllable production of nanometer selenium：Seed culture fluid is seeded in 1L triangular flask fermentation mediums according to 5% inoculum concentration, filled
Liquid measure is 400mL, and fermentation adds 500mg/L sodium selenite before starting, and adds 250mg/L sodium selenite after 24h, 25 DEG C
80r/min Shaking cultures 48h.
Nanometer selenium extraction purification：By zymotic fluid 10000r/min refrigerated centrifuge 10min, collection obtains thalline, with 100mL without
Bacterium normal saline flushing three times, is suspended with 20mL volume sterile distilled waters, carries out ice-bath ultrasonic broken wall, 30W, start and stop interval
15s, 20min, cellular lysate liquid 10000r/min refrigerated centrifuge 10min being crushed, precipitation is cleaned three times with 20mL deionized waters, and
Aqueous suspension is distilled with 10mL volumes, obtains nanometer selenium suspension, same volume chloroform is added and carries out extraction 30min, extract 3 times, merge
Lower floor's aqueous phase, with homogenizer homogeneous 5min, 10000r/min refrigerated centrifuge 10min, precipitation cleans 3 with 10mL sterile salines
Secondary, freeze-drying 24h obtains microorganism nanometer selenium.
The detection of the purity and average grain diameter of nanometer selenium：The detection of selenium is carried out according to standard GB/T 5009.93-2017,
On this condition, the conversion ratio of nanometer selenium is 88.31%, and the purity of gained selenium is 91.2%, and average grain diameter is 153 ± 20nm.
Embodiment 5：It is prepared by nanometer selenium
Actication of culture：Preservation strain is connected on beef extract-peptone test tube agar slant culture-medium, 40 DEG C of static gas wave refrigerators
Activation, treats that bacterium colony covers with inclined-plane.
Seed culture：Inclined-plane culture strain is inoculated in into the beef extract-peptone liquid containing 100mg/L sodium selenites to train
Support in base, liquid amount is the 20% of container capacity, 40 DEG C, 200r/min shaken cultivation 24h, obtains seed liquor.
The controllable production of nanometer selenium：Seed culture fluid is seeded in 500L ferment tank culture mediums according to 5% inoculum concentration,
Liquid amount is 350L, and by the way of continuous stream plus sodium selenite mother liquor (50g/L), flow acceleration 1.5mL/min, continuous stream adds
36h, 40 DEG C of 200r/min fermented and cultureds 72h.
Nanometer selenium extraction purification：By ferment tank liquid 4000r/min refrigerated centrifuge 20min, collection obtains thalline, uses
100L sterile salines flush three times, and are suspended with 15L volume sterile distilled waters, carry out ice-bath ultrasonic broken wall, 800W, open
Stop being spaced 15s, crush 20min, cellular lysate liquid 5000r/min refrigerated centrifuge 20min, precipitation cleans three with 15L deionized waters
It is secondary, and aqueous suspension is distilled with 5L volumes, nanometer selenium suspension is obtained, same volume chloroform is added and carries out extraction 30min, extract 3 times, close
And lower floor's aqueous phase, with homogenizer homogeneous 5min, 5000r/min refrigerated centrifuge 10min, precipitation cleans 3 with 5L sterile salines
Secondary, freeze-drying 48h obtains microorganism nanometer selenium.
The detection of the purity and average grain diameter of nanometer selenium：The detection of selenium is carried out according to standard GB/T 5009.93-2017；
The conversion ratio of nanometer selenium is 94.32% on this condition, and the purity of gained selenium is 91.6%, and average grain diameter is 116 ± 20nm.
Embodiment 6：It is prepared by nanometer selenium
Actication of culture：Preservation strain is connected on beef extract-peptone test tube agar slant culture-medium, 30 DEG C of static gas wave refrigerators
Activation, treats that bacterium colony covers with inclined-plane.
Seed culture：Inclined-plane culture strain is inoculated in into the beef extract-peptone liquid containing 100mg/L sodium selenites to train
Support in base, liquid amount is the 20% of container capacity, 30 DEG C, 140r/min shaken cultivation 24h, obtains seed liquor.
The controllable production of nanometer selenium：Seed culture fluid is seeded to 100L small-sized fermentation tank fermented and cultureds according to 5% inoculum concentration
In base, liquid amount 50L, fermentation adds 500mg/L sodium selenite before starting, and continuous stream is carried out after 24h and adds sodium selenite female
Liquid (50g/L), 0.1mL/min, continuous stream add 24h, and 30 DEG C of 140r/min stirrings co-culture 60h.
Nanometer selenium extraction purification：By zymotic fluid 8000r/min refrigerated centrifuge 15min, collection obtains thalline, sterile with 10L
Normal saline flushing three times, is suspended with 2L volume sterile distilled waters, carries out ice-bath ultrasonic broken wall, 600W, start and stop interval
15s, crushes 20min, cellular lysate liquid 8000r/min refrigerated centrifuge 15min, and precipitation is cleaned three times with 1L deionized waters, is used in combination
1L volumes distill aqueous suspension, obtain nanometer selenium suspension, add same volume chloroform and carry out extraction 30min, extract 3 times, merge lower floor
Aqueous phase, with homogenizer homogeneous 5min, 8000r/min refrigerated centrifuge 15min, precipitation is cleaned 3 times with 1L sterile salines, is freezed
Dry 48h and obtain microorganism nanometer selenium.
The detection of the purity and average grain diameter of nanometer selenium：The detection of selenium is carried out according to standard GB/T 5009.93-2017,
The conversion ratio of nanometer selenium is 91.3% on this condition, and the purity of gained selenium is 92%, and average grain diameter is 145 ± 20nm.
Embodiment 7：It is prepared by nanometer selenium
Actication of culture：Preservation strain is connected on beef extract-peptone test tube agar slant culture-medium, 35 DEG C of static gas wave refrigerators
Activation, treats that bacterium colony covers with inclined-plane.
Seed culture：Inclined-plane culture strain is inoculated in into the beef extract-peptone liquid containing 100mg/L sodium selenites to train
Support in base, liquid amount is the 20% of container capacity, 35 DEG C, 180r/min shaken cultivation 24h, obtains seed liquor.
The controllable production of nanometer selenium：Seed culture fluid is seeded in 1T ferment tank culture mediums according to 5% inoculum concentration, filled
Liquid measure is 600L, and by the way of continuous stream plus sodium selenite mother liquor (50g/L), flow acceleration 2mL/min, continuous stream adds 48h
Afterwards, flow velocity is changed into 1ml/min, stream plus 12h, 35 DEG C of 180r/min common fermentation cultures 72h.
Nanometer selenium extraction purification：By zymotic fluid 5000r/min refrigerated centrifuge 20min, collection obtains thalline, sterile with 150L
Normal saline flushing three times, is suspended with 30L sterile distilled waters, progress ice-bath ultrasonic broken wall, 1000W, start and stop interval 15s,
Broken 20min, cellular lysate liquid 5000r/min refrigerated centrifuge 20min, precipitation is cleaned three times with 15L deionized waters, and uses 15L
Volume distills aqueous suspension, obtains nanometer selenium suspension, adds same volume chloroform and carries out extraction 30min, extract 3 times, merges lower floor's water
Phase, with homogenizer homogeneous 5min, 5000r/min refrigerated centrifuge 20min, precipitation is cleaned 3 times with 15L sterile salines, is freezed
Dry 48h and obtain microorganism nanometer selenium.
The detection of the purity and average grain diameter of nanometer selenium：The detection of selenium is carried out according to standard GB/T 5009.93-2017,
On this condition, the conversion ratio of nanometer selenium is 90.8%, and the purity of gained selenium is 93.2%, and average grain diameter is 153 ± 20nm.
Use above specific case is illustrated to the present invention, is only intended to help and is understood the present invention, not limiting
The system present invention.For those skilled in the art, according to the thought of the present invention, can also make some simple
Deduce, deform or replace.
SEQUENCE LISTING
<110>Shaanxi Institute of Microbiology
<120>A kind of bacillus amyloliquefaciens Lxz-41 and the method using the bacterial strain controllable preparation nanometer selenium
<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 kind of bacillus amyloliquefaciens (Baci l lus amylol iquefaciens) Lxz-41, deposit number are：
CCTCC M2016578, are deposited in China typical culture collection center, and the bacterial strain has the ability of controllable production nanometer selenium.
2. a kind of bacillus amyloliquefaciens (Baci l lus amylol iquefaciens) controllable production nanometer seleniums of Lxz-41
Method, it is characterised in that comprise the steps of:
S1. actication of culture：Preservation strain is connected on beef extract-peptone or LB test tube agar slant culture-mediums, 25-40 DEG C quiet
Only culture activation, treats that bacterium colony covers with inclined-plane；
S2. seed culture：Inclined-plane culture strain is inoculated in the beef extract-peptone fluid nutrient medium containing selenium salt, liquid amount
For 20%, 25-40 DEG C of container capacity, 80-200r/min shaken cultivation 12-24h, seed liquor is obtained；
S3. the controllable production of nanometer selenium：Seed culture fluid is seeded to the fermentation equipped with fermentation medium according to 1%-5% inoculum concentrations
In container, the fermentation medium includes selenium salt, and selenium salt is added using stream plus form, and fermentation medium liquid amount holds for fermentation
The 40%-70% of device capacity, stir speed (S.S.) 50-200r/min, throughput 0.5-2.0vvm, temperature are kept for 25-40 DEG C, during fermentation
Between 24-72h；
S4. nanometer selenium extraction purification：Tank under zymotic fluid, 4000-10000r/min4 DEG C of refrigerated centrifuge 10-20min, collection obtain
Thalline, flushed three times with about 1/4 fermentating liquid volume sterile saline, suspended, entered with about 1/20 volume sterile distilled water
Row ice-bath ultrasonic broken wall 20-40min, 4000-10000r/min4 DEG C of refrigerated centrifuge 10-20min of cellular lysate liquid, precipitation is with together
Volumes of deionized water is cleaned three times, and distills aqueous suspension with about 1/2 volume, obtains nanometer selenium suspension, is added same volume chloroform and is entered
Row extraction 30min, extracts 2-3 times, merges lower floor's aqueous phase, with homogenizer homogeneous 2-5min, 4000-10000r/min4 DEG C of freezing
10-20min is centrifuged, precipitation is cleaned 3 times with isometric sterile saline, is freeze-dried and produces microorganism nanometer selenium.
3. according to the method for claim 2, it is characterised in that the selenium salt in S2 in beef extract-peptone fluid nutrient medium is
One or more in selenite, selenate, mass volume ratio 100-500mg/L.
4. according to the method for claim 2, it is characterised in that the selenium salt in S2, S3 culture medium is individually sterilized, so
It is added to afterwards in the culture medium after sterilizing.
5. according to the method for claim 2, it is characterised in that fermentation medium component is (g/L) in S3：Glucose 10,
Peptone 5, ammonium sulfate 1, epsom salt 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, pH=7.2-7.4.
6. according to the method for claim 5, it is characterised in that the selenium salt in S3 in fermentation medium is flowed in batches with mother liquor to be added
Form or continuous stream add form, to control the concentration of selenium salt in zymotic fluid, and then control the transformation efficiency of bacterial strain, the selenium salt is
One or more in selenite, selenate.
7. according to the method for claim 6, it is characterised in that stream adds in batches in fermentation medium or continuous stream adds selenous acid
Sodium, the concentration of sodium selenite mother liquor used is 50-500g/L.
8. according to the method for claim 2, it is characterised in that ultrasonication power 400W-1000W, frequency 20- in S4
40KHz, start and stop interval 10-15s, crush 20-40min.
9. according to the method for claim 5, it is characterised in that the surfactant is SDS, polysorbate40, polysorbate60, told
One or more in temperature 80.
10. the complex micro organism fungicide containing bacillus amyloliquefaciens Lxz-41 described in claim 1.
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| CN113373180A (en) * | 2020-12-18 | 2021-09-10 | 中国农业科学院烟草研究所 | Nano-selenium synthetic active bacterial liquid of bacillus amyloliquefaciens, 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 |
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| CN111235066A (en) * | 2020-03-12 | 2020-06-05 | 陕西省微生物研究所 | Bacillus belgii Lxh and method for preparing agricultural nano-selenium tablet by using same |
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| CN113373180A (en) * | 2020-12-18 | 2021-09-10 | 中国农业科学院烟草研究所 | Nano-selenium synthetic active bacterial liquid of bacillus amyloliquefaciens, preparation method and application thereof |
| 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 |
| CN115851544A (en) * | 2022-12-16 | 2023-03-28 | 陕西省微生物研究所 | Bacillus subtilis and method for preparing selenium-rich natto by using same |
| 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 |
| CN115927114A (en) * | 2022-12-27 | 2023-04-07 | 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, and method and application thereof for synthesizing nano-selenium |
| 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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