CN115678793A - Bacillus subtilis subspecies natto N14 and application thereof - Google Patents
Bacillus subtilis subspecies natto N14 and application thereof Download PDFInfo
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Abstract
The invention discloses a bacillus subtilis subspecies natto N14 and application thereof. The invention provides bacillus subtilis natto subspecies N14 (namely natto bacterium N14), which can tolerate selenite with high concentration and is suitable for producing biological nano selenium and organic selenium by large-scale fermentation. The bacillus subtilis natto subspecies N14 strain can efficiently synthesize organic selenium (selenomethylselenocysteine and selenomethionine), and the bacillus natto N14 can also be used for producing selenium-rich soybeans with high content of selenomethionine; further utilizing the strain N14 to carry out solid state fermentation to completely convert inorganic selenium in the selenium-rich soybeans into selenomethionine organic selenium. The selenium-rich natto with 100 percent of organic selenium content is obtained, and meanwhile, the yield of gamma-polyglutamic acid and nattokinase is not obviously influenced.
Description
Technical Field
The invention relates to the technical field of biological nano-selenium preparation, in particular to bacillus subtilis and subspecies natto N14 and application thereof.
Background
Selenium (Selenium) is a trace element essential to human and animals, participates in the synthesis of various Selenium-containing proteins such as glutathione peroxidase in the form of selenocysteine, and has important effects in improving organism immunity, resisting oxidation, protecting liver, kidney and cardiovascular, preventing diabetes and thyroid diseases, resisting virus and cancer, etc. Also, selenium has a very narrow range of toxicity and activity, and excessive ingestion can lead to intoxication including demethylation, hair loss, staggering, etc.
Selenium compounds with different forms have obviously different toxicity and activity characteristics, compared with inorganic selenium such as selenite, selenate and the like, the bioavailability of organic selenium such as L-selenium-methyl selenocysteine, selenomethionine and the like is higher, and the Caco-cell model result shows that the bioavailability of SeMet (56 +/-4%) > selenium methyl selenocysteine (46 +/-2%) > Se (VI) (33 +/-2%) > Se (IV) (12 +/-1%) (Thiry et al.2013). Compared with other organic selenium, the selenium methyl selenocysteine has good anticancer prospect, and is a methylated derivative of the 21 st human body essential amino acid selenocysteine. The selenium methyl selenocysteine has the functions of supplementing selenium, preventing and treating cancers, resisting oxidation and aging, treating cardiovascular and cerebrovascular diseases, removing heavy metal toxicity and the like, and is approved by the Ministry of health in 2009 to be a novel nutrition enhancer. The selenium-supplementing nutrition enhancer and the broad-spectrum anticancer agent play important roles in the aspects of food, medical health-care products and the like. However, the selenium methyl selenocysteine is mainly synthesized chemically at present, and only reports of edible fungi hypsizygus marmoreus are found in the aspect of synthesizing high-efficiency biological selenium methyl selenocysteine, and reports of high-efficiency synthesis of selenium methyl selenocysteine bacterial strains are not found. The nano selenium (elemental selenium with the nano scale) has obvious biological activity, has lower toxicity than inorganic selenium and organic selenium, is a novel safe and efficient selenium supplement, and shows multiple biological activities in the aspects of antibiosis, antivirus, antitumor and the like.
The nano selenium can be prepared by a chemical or biological synthesis method, wherein biological macromolecules such as protein, polysaccharide and the like are coated on the surface of the biological synthesized nano selenium, so that the nano selenium has better stability and is not easy to be converted into inactive crystalline elemental selenium. Various microorganisms have been found to be capable of reducing inorganic selenium to synthesize biological nano-selenium, including Aeromonas (Aeromonas) of proteobacteria, agrobacterium (Agrobacterium), pseudomonas (Pseudomonas), bacillus (Bacillus) of firmicutes, lactobacillus (Lactobacillus), lactococcus (Lactococcus), staphylococcus (Staphylococcus), and frank (Frankia), streptomyces (Streptomyces) of actinomycetes, and the like. The safe and efficient bacterial strain is the first problem to be solved in the biological nano-selenium industrialization process, the bacterial strain for fermentation production must meet the requirements of safety, high efficiency and easy culture, has no safety risk to the environment and organisms, has high conversion rate and high synthesis rate, is easy to amplify and culture in the fermentation production, and the synthesized biological nano-selenium has good stability and does not denature and inactivate in the processing process.
Bacillus subtilis natto (Bacillus subtilis natto) is also called natto bacteria, is a fermentation strain of a traditional Japanese food natto, has more than thousand years of eating history, is rich in various active substances such as gamma-polyglutamic acid, nattokinase, vitamin K and the like, and is considered as an important diet habit for prolonging the life of people in Japan; in China, more than two thousand years of history of eating fermented soybeans, fermented soybeans contain abundant flora such as bacillus besides aspergillus and microzyme. Bacillus natto is a functional strain which is generally recognized as safe.
Disclosure of Invention
The invention aims to provide a bacillus subtilis natto subspecies N14 (bacillus natto N14) which can be used for food, has strong inorganic selenium tolerance, high biological nano selenium yield and uniform particle size, can efficiently synthesize L-selenium-methyl selenocysteine and selenomethionine, and can efficiently synthesize nattokinase and gamma-polyglutamic acid.
The invention also aims to provide the application of the bacillus natto N14 in the production of biological nano-selenium and the production of selenium-rich plants.
In order to realize the purpose of the invention, in the first aspect, the invention provides a Bacillus subtilis natto subsp.natto N14 separated from fermented soya beans, wherein the strain N14 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the address of No. 3 North Chen Xilu No. 1 of the Chaoyang district of Beijing City, the microbial research institute of Chinese academy of sciences, the postal code 100101, the preservation number CGMCC No.21703, and the preservation date of 2021 year, 1 month and 22 days.
The 16S rRNA gene and the gyrB gene of the strain N14 are respectively shown in SEQ ID NO 1-2.
In a second aspect, the present invention provides a microbial preparation comprising Bacillus natto N14.
In a third aspect, the invention provides a method for biosynthesizing nano-selenium by using bacillus natto N14, which comprises the following steps: adding selenite (such as sodium selenite) with final concentration of 1-100mM to the fermentation medium, fermenting and culturing strain N14, and separating and purifying nano selenium from the fermentation product.
The nano selenium has the particle size of 100-200 nm and is in a spherical amorphous state.
In a fourth aspect, the invention provides a method for producing organic selenium by using bacillus natto N14, which comprises the following steps: adding selenite (such as sodium selenite) with final concentration of 0.01-0.1mM to the fermentation medium, fermenting and culturing strain N14, and separating and purifying organic selenium (including selenomethylselenocysteine and selenomethionine) from the fermentation product.
In the aforementioned method, the formulation of the fermentation medium (NT 4) is: 18-24g/L of glucose, 1.8-2.6g/L of glycerol, 4-6g/L of yeast extract, 4.2-5.8g/L of peptone, 8.2-12.6g/L of L-sodium glutamate, 0.2-0.3g/L of magnesium sulfate heptahydrate, 2.3-3.6g/L of dipotassium phosphate and 7.0-7.5 of pH.
In one embodiment of the invention, the method for biologically synthesizing nano-selenium by using the bacillus natto N14 in the 1000L fermentation tank is as follows:
1. strain activation
Taking N14 glycerol tube strain (2 mL) stored at-80 ℃, thawing at room temperature, inoculating to 50mL LB shake flask, and performing shake culture at 37 ℃ and 180rpm for 16h for activation.
2. Shake flask seed preparation
20mL of the activated bacterial solution was transferred to 1000mL of shake flask (seed medium) and subjected to shaking culture at 37 ℃ and 180rpm for 6 hours to serve as shake flask seeds.
3. Seeding tank culture
Inoculating 700mL shake flask seed solution into 50L seed tank (seed culture medium containing 35L), controlling fermentation temperature at 37 deg.C, stirring at 150rpm, and ventilating at 2m 3 And h, fermenting for 8h.
The formula of the seed culture medium is as follows: 20g/L glucose, 5g/L yeast extract, 5g/L soyabean peptone, 0.2g/L magnesium sulfate heptahydrate, 2g/L dipotassium hydrogen phosphate, pH 7.0, sterilizing at 121 ℃ for 20min.
4. Fermentation of nano-selenium
Adding 700L fermentation medium NT4 into 1000L fermentation tank, sterilizing at 121 deg.C for 20min, cooling to about 37 deg.C, transferring into 35L seed tank, controlling fermentation temperature at 37 deg.C, stirring at 150rpm, and ventilation amount of 42m 3 And/h, the fermentation time is 30h, and selenite is supplemented into the fermentation tank by constant-speed feeding (the concentration is 1M, the autoclaving at 121 ℃ is 20min, the feeding rate is 10mL/min, and the total amount is 7L and about 12 h). And measuring the biological nano-selenium content of the fermentation liquor.
In a fifth aspect, the invention provides an application of bacillus natto N14 in preparing selenium-rich plants (such as selenium-rich soybeans).
In a sixth aspect, the invention provides a production method of selenium-rich soybeans, which comprises the following steps:
A. adding selenite with the concentration of 1-100mM into a fermentation culture medium, and fermenting and culturing the bacillus natto N14;
B. and (4) applying the fermentation liquid obtained in the step A to soil around the rhizosphere of soybean plants or spraying the fermentation liquid to leaf surfaces, and harvesting after the soybeans are mature.
The obtained selenium-rich soybean contains total selenium 0.64-3.49mg/kg, organic selenium 85.0-96.3%, and tetravalent selenium in balance.
In the present invention, the ratio of the organic selenium means mass percentage, i.e. the content of the organic selenium (in terms of selenium) is divided by the total selenium content (in terms of selenium).
In a seventh aspect, the invention provides a method for producing selenium-rich natto, which comprises the following steps:
1) Soaking the selenium-rich soybeans prepared by the method in water, and steaming the soaked soybeans in a water-proof way;
2) Cooling the cooked soybean, inoculating bacillus natto N14, fermenting and after-ripening to obtain the selenium-rich natto.
Preferably, the fermentation conditions are: fermenting in an incubator at 37 ℃ for 24h.
Preferably, the after-ripening conditions are: after-ripening for 24h at 4 ℃.
The obtained selenium-rich natto contains total selenium 0.66-3.83mg/kg, organic selenium 100%, nattokinase 174.2-196.9FU/g, and gamma-polyglutamic acid 158.5-164.0mg/g.
The invention provides another production method of selenium-rich natto, which comprises the following steps:
(1) Soaking semen glycines in water containing selenite;
(2) Steaming the soaked soybeans in a water-proof way;
(3) Cooling the cooked soybeans, inoculating bacillus natto N14, fermenting and after-ripening to obtain the selenium-rich natto.
Preferably, the fermentation conditions are: fermenting in an incubator at 37 ℃ for 24h.
Preferably, the after-ripening conditions: after-ripening for 24h at 4 ℃.
The obtained selenium-rich natto contains total selenium 0.95-6.42mg/kg, organic selenium (SeMet) 60.5-82.2%, nattokinase 156.4-234.2FU/g, and gamma-polyglutamic acid 149.2-188.0mg/g.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the bacillus natto N14 disclosed by the invention can tolerate selenite with high concentration (can tolerate selenite with the concentration of 100mM in a fermentation culture solution), and is suitable for large-scale fermentation production of biological nano-selenium. The Bacillus natto N14 can be used for producing selenium-rich soybean with high content of selenomethionine; further utilizing the strain N14 to carry out solid state fermentation to completely convert inorganic selenium in the selenium-enriched soybeans into selenomethionine organic selenium. The selenium-rich natto with 100 percent of organic selenium content is obtained, and meanwhile, the yield of gamma-polyglutamic acid (gamma-PGA) and nattokinase is not obviously influenced.
Drawings
FIG. 1 shows the evolved trees of the 16S rRNA (A) and gyrB genes (B) of strain N14 and fermented natto (C) in the preferred embodiment of the present invention.
FIG. 2 shows the biological nano-selenium production (A) and conversion (B) of strain N14 at 0-100mM sodium selenite concentration in the preferred embodiment of the present invention.
FIG. 3 shows the characteristics of the N14 bacteria and the bio-nano-selenium under the TEM of the preferred embodiment of the present invention. Wherein A is a control group, B is a 5mM sodium selenite treated group, and C is purified nanoparticles.
FIG. 4 is a selected area electron diffraction (A) and energy spectrum (B) of the strain N14 biological nano selenium in the preferred embodiment of the invention.
FIG. 5 is a graph showing the synthesis of bacterial strain N14 at 5, 10 and 20mM sodium selenite concentrations in the presence of bioselenium in a preferred embodiment of the present invention.
FIG. 6 is a photograph (A) and a photograph (B) showing the production of biological nanoselenium by strain N14 in a 1000L fermentor in accordance with a preferred embodiment of the present invention.
FIG. 7 shows the selenium form of N14 bacteria in the preferred embodiment of the present invention. Wherein, A is 5 kinds of selenium form standard samples, and B is N14 thallus selenium form.
FIG. 8 is a graph showing the effect of selenate and selenite on the synthesis of γ -PGA from N14 according to a preferred embodiment of the present invention.
FIG. 9 shows the selenium content of normal and selenium-rich soybeans, normal and selenium-rich natto in a preferred embodiment of the present invention.
FIG. 10 shows the selenium form of the selenium-enriched soybean 3 and the selenium-enriched natto 3 according to the preferred embodiment of the present invention.
FIG. 11 shows the gamma-PGA and nattokinase contents of ordinary and selenium-enriched natto in a preferred embodiment of the present invention.
FIG. 12 is a graph showing the resistance of the Bacillus natto N14 solid plates to selenite in a preferred embodiment of the present invention.
FIG. 13 is a graph showing the resistance of Bacillus natto N14 to selenite under liquid culture conditions in a preferred embodiment of the present invention.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available.
Example 1 isolation and identification of Bacillus natto N14
Collecting 12 fermented soybean product fermented soybeans from Henan, shandong and Hebei, separating to obtain 16 bacillus strains (N1-N16) together, adding sodium selenite into LB culture medium to a final concentration of 5mM,10mM and 2mM, and detecting the tolerance capacity of the 16 strains to the sodium selenite, wherein the highest tolerance concentration of N2, N3, N4, N6, N7, N10, N11, N12, N13 and N16 to the sodium selenite is 5mM, and the highest tolerance concentration of N1, N5, N8, N9 and N15 to the sodium selenite is 10mM, and only N14 can grow well on the LB culture medium with the sodium selenite concentration of 20mM, which indicates that the tolerance concentration of N14 to the sodium selenite exceeds 20mM, and carrying out further subsequent research on the N14 strain.
After N14 strain genome DNA was extracted, the 16S rRNA gene (SEQ ID NO: 1) was amplified using 27F (5 '-AGAGAGTTTGATCCTGGCTCAG-3') and 1492R (5 '-GGTTACCTTGTTACGACTT-3') as primers; the gyrB gene (SEQ ID NO: 2) was amplified using UP1f (5 'GAAGTCATCATCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3') and UP2r (5 'AGCAGGGTACGGATGTGCGAGCCTCNARTCNGCRTCNGTCAT-3') as primers.
PCR products were purified and sequenced, the sequencing results were spliced using DNAMAN software, and similarity comparisons were performed with bacteria in the NCBI database (https:// BLAST. NCBI. Nlm. Nih. Gov/BLAST. Cgi) using the BLAST program. The result shows that the consistency of the 16S rRNA gene sequence of the N14 strain and Bacillus subtilis reaches 99.93 percent; the consistency of the gyrA gene sequence and Bacillus subtilis reaches 100 percent, and the consistency of the gyrA gene sequence and two strains of Bacillus natto CGMCC 2108 and BEST195 reaches 100 percent. The 16S rRNA and gyrB gene evolutionary trees of the N14 strain are shown in A and B in FIG. 1. The natto obtained by fermenting with N14 is shown in figure 1C, and is light yellow, has special flavor and taste of natto, no foreign odor, high mucus content, strong viscosity, good wire drawing state, and moderate softness and hardness. Therefore, N14 can be determined as Bacillus subtilis natto (natto), and the strain N14 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO.21703.
Example 2 biological Nano-selenium production and conversion of Bacillus natto N14 at 0.01 to 100mM selenite concentration
An N14 single colony is picked and inoculated into an LB test tube, after 12h of shaking culture at 37 ℃ and 150rpm, 1mL of the single colony is taken to be transferred into a 50mL LB shaking flask, and the single colony is shaken at 37 ℃ and 150rpm for 5h to be used as seed liquid. 200 mu L of seed solution and a corresponding volume of sodium selenite mother solution (1M, filtration and sterilization) are inoculated in a test tube filled with 5mL of biological nano-selenium fermentation medium NT4 (20 g/L of glucose, 2g/L of glycerol, 5g/L of yeast extract, 5g/L of peptone, 10g/L of L-sodium glutamate, 0.2g/L of magnesium sulfate heptahydrate, 2g/L of dipotassium phosphate, pH 7.0 and sterilization at 121 ℃ for 20 min) so that the concentrations of selenite are respectively 0, 0.01, 0.1, 1, 5, 10, 20, 40, 60, 80 and 100mM, and the yield of the biological nano-selenium is measured after the culture is placed at 37 ℃ and 150rpm and shaking culture is carried out for 48h.
The yield of the biological nano-selenium is measured by adopting a sodium sulfide spectrophotometry. Preparation of Na from ultrapure Water 2 S solution (1M, ready for use), taking 500 mu L fermentation liquor to be tested, centrifuging at 12000rpm for 5min, discarding the supernatant, washing with physiological saline for 3 times, discarding the supernatant, adding 1mL Na 2 And (3) fully and uniformly mixing the S solution, reacting for 1h, centrifuging at 12000rpm for 5min, taking the supernatant, measuring the absorbance at the wavelength of 500nm, and calculating the yield and the conversion rate of the biological nano-selenium in the fermentation liquor according to a nano-selenium standard curve.
TEM electron microscopy and EDX spectroscopy. Centrifuging fermentation liquor of a control group and fermentation liquor of a 5mM selenite treatment group at 1 mL/4000rpm for 10min respectively to collect precipitates, washing the precipitates for 3 times by using normal saline and suspending the precipitates, dropwise adding a drop of suspension liquid on a carbon-supported membrane copper net, absorbing excessive water by using filter paper, airing the precipitates, and observing thalli and nanoparticles under a transmission electron microscope; and (3) carrying out appearance observation on the selected area nano particles under a high-resolution transmission electron microscope, analyzing the crystal form characteristics of the nano particles by combining an electron diffraction structure, and carrying out element composition analysis on the nano particles by using an energy spectrum analyzer (EDX).
The results show that in the biological nano-selenium fermentation culture solution, N14 can tolerate selenite with the content of 100mM, and reduce selenite to synthesize biological nano-selenium under the condition of 1-100mM of initial selenite addition, so that the culture solution presents bright red, and the culture solution does not produce red under the conditions of 0.01 and 0.1mM of selenite concentration. Wherein, the biological nano-selenium yield is highest at 60mM and 40mM selenite concentrations, respectively reaching 18.04mM (1.42 g/L) and 16.82 mM (1.33 g/L), while the biological nano-selenium conversion rate is highest at 5mM and 10mM, reaching 89.62% and 87.53% (FIG. 2, A and B).
The cell membranes of 5mM selenite-treated cells and a large number of uniform nanospheres with particle sizes ranging from 100 nm to 200nm (see FIGS. 3, A-C) were observed under a transmission electron microscope. FIG. 4 (A and B) Selective Electron diffraction (SEAD) of nanoparticles shows diffuse diffraction rings, indicating that the crystalline form of the nanoparticles is amorphous; the energy spectrum shows specific absorption peaks of elemental selenium at 1.38 KeV, 11.22KeV and 12.50KeV, indicating that the amorphous nanoparticles are nano-selenium.
Example 3 Synthesis Curve of biological Nano-selenium with Bacillus natto N14 at 5-20 mM selenite concentration
The single colony of N14 is selected and inoculated into an LB test tube, after 12h of shaking culture at 37 ℃ and 150rpm, 1mL of the single colony is taken to be transferred into a 50mL LB shaking flask, and 5h of shaking culture at 37 ℃ and 150rpm is taken as seed liquid. Transferring 2mL of the seed solution into a 500mL conical flask containing 100mL of biological nano-selenium fermentation medium NT4 (glucose 18g/L, glycerol 2.6g/L, yeast extract 6g/L, peptone 4.2g/L, L-sodium glutamate 8.2g/L, magnesium sulfate heptahydrate 0.3g/L, dipotassium hydrogen phosphate 3.6g/L, pH 7.2), adding corresponding volumes of sodium selenite solution (1M, filtering and sterilizing) to make the final concentrations of sodium selenite be 5, 10 and 20mM respectively, performing shake culture at 37 ℃ and 150rpm for 48h, and periodically sampling to determine the biological nano-selenium content in the culture solution.
According to Na 2 The S spectrophotometer measures the yield of the biological nano-selenium in the fermentation liquor and calculates the conversion rate, and the result is shown in figure 5. Culturing in N14In the early stage (8-12 h), the yield of biological nano-selenium is increased rapidly, and then slowly increased (10 mM and 20mM treatment groups) or stabilized (5 mM treatment group). The yield of biological nano-selenium reaches 4mM (the conversion rate is 80%) after 8 hours of culture under the condition of 5mM of initial selenite concentration, and the conversion rate reaches 90% (4.5 mM) after 18 hours; under the condition of 10mM selenite concentration, the yield of the biological nano-selenium reaches 7.5mM (the conversion rate is 75%) at 24h, and is increased to 8.4mM (the conversion rate is 84%) after 48 h; and under the concentration of 20mM selenite, the yield of the biological nano-selenium reaches 9.2mM (the conversion rate is 46%) at 24h, and is increased to 11.6mM (the conversion rate is 58%) after 48h.
Example 4 method for biosynthesis of Nano selenium from Natto bacterium N14 in 1000L fermenter
1. Bacterial activation
Taking N14 glycerol tube strain (2 mL) stored at-80 ℃, thawing at room temperature, inoculating to a 50mL LB shake flask, and performing shaking culture at 37 ℃ and 180rpm for 16h for activation.
2. Shake flask seed preparation
20mL of the activated bacterial solution was transferred to 1000mL of a shake flask (seed medium) and subjected to shaking culture at 37 ℃ and 180rpm for 6 hours to serve as a shake flask seed.
3. Seeding tank culture
Inoculating 700mL shake flask seed solution into 50L seed tank (seed culture medium containing 35L), controlling fermentation temperature at 37 deg.C, stirring at 150rpm, and ventilating at 2m 3 And h, fermenting for 8h.
The formula of the seed culture medium is as follows: 20g/L of glucose, 5g/L of yeast extract, 5g/L of soybean peptone, 0.2g/L of magnesium sulfate heptahydrate, 2g/L of dipotassium phosphate, pH 7.0 and sterilizing at 121 ℃ for 20min.
4. Fermentation of nano-selenium
Putting 700L biological nano selenium fermentation medium NT4 (glucose 24g/L, glycerol 1.8 g/L, yeast extract 4g/L, peptone 5.8g/L, L-sodium glutamate 12.6g/L, magnesium sulfate heptahydrate 0.2g/L, dipotassium hydrogen phosphate 2.3g/L, pH 7.2) into 1000L fermentation tank, sterilizing at 121 deg.C for 20min, cooling to about 37 deg.C, transferring to 35L seed tank, controlling fermentation temperature at 37 deg.C, stirring at 150rpm, and ventilation amount of 42m 3 The fermentation time is 30 hours, and selenite is added into the fermentation tank in a constant-speed feeding manner (the concentration is 1M,autoclaving at 121 deg.C for 20min at a feeding rate of 10mL/min for 7L about 12 h). Na for timed sampling 2 And (3) measuring the biological nano-selenium content of the fermentation liquor by an S spectrophotometry.
The yield of biological nano-selenium in the fermentation liquid is shown in figure 6 (A and B), under the condition of 10mM of total sodium selenite fed-batch, after fermentation is carried out for 18h in a 1000L fermentation tank, the conversion rate of the biological nano-selenium reaches more than 90% (9.2 mM), the reduction rate is faster than that of a shake flask, and the yield is 9.7mM when the fermentation is carried out for 30 h.
Example 5 Synthesis of organic selenium by conversion of selenite with Bacillus natto N14
After N14 single colonies were picked and inoculated into LB tubes, shaken at 37 ℃ and 150rpm for 12 hours for activation, 1mL was transferred to 100mL NT4 medium shake flasks, and sodium selenite solution (1M, filter sterilized) was added to give a final concentration of 0.1mM sodium selenite, and shaken at 37 ℃ and 150rpm for 48 hours.
Selenium morphology analysis was performed using a morphology analyzer (HPLC-HG-AFS). Centrifuging the bacterial liquid at 12000rpm for 5min, washing with ultrapure water for 3 times, adding 20mg/mL lysozyme solution for resuspension, ultrasonically crushing, adding protease 14 solution (final concentration of 8 mg/mL) and placing in a shaking table at 37 ℃, shaking for enzymolysis for 12h, centrifuging at 12000rpm for 5min, filtering the supernatant with a 0.22 mu m filter membrane, and detecting the selenium form, wherein the measurement parameters of morphological analysis are shown in Table 1.
TABLE 1HPLC-HG-AFS determination of the Instrument Condition parameters
The results are shown in FIG. 7 (A and B), N14 can reduce selenite into two organic selenides of L-selenium-methyl selenocysteine (MeSeCys, 19%) and selenomethionine (SeMet, 49%), the total selenium content of the thallus reaches 1.4 +/-0.2 mg/g (dry weight), and the selenium enrichment efficiency is 18%.
Example 6 Effect of selenium on the Synthesis of γ -PGA by Bacillus natto N14
Selecting N14 single colony, inoculating into LB test tube, shaking at 37 deg.C and 150rpm for 12 hr for activation, taking 1mL, transferring into 50mL LB shake flask, shaking at 37 deg.C and 150rpm for 5 hr, and adjusting OD of bacterial liquid 600 =0.8 as seed liquid. Inoculating the strain to gamma-PGA according to 2 percent of inoculation amountAdding filter sterilized sodium selenate or sodium selenite solution into the culture medium to make Se (VI) content of the culture medium 1, 10, 50mg/L, se (IV) content 0.1, 0.5, 1mg/L, respectively, shaking at 37 deg.C and 180rpm for 48h.
γ -PGA Synthesis Medium: glucose 30g/L, glycerol 5g/L, NH 4 Cl 5g/L, L-sodium glutamate 20g/L, K 2 HPO 4 2g/L，MgSO 4 ·7H 2 O 0.2g/L，MnSO 4 0.05g/L，CaCl 2 0.1g/L, pH 7.0, sterilizing at 121 deg.C for 20min.
The content of the gamma-PGA in the bacterial liquid is measured by an ultraviolet spectrophotometry. Diluting the bacterial liquid by 5 times, centrifuging at 12000rpm for 10min, taking 0.3mL of supernatant, adding 1.2mL of glacial ethanol to precipitate gamma-PGA, centrifuging at 12000rpm for 10min, discarding the supernatant, drying the precipitate in vacuum, and redissolving in 1.2mL of ultrapure water. Centrifuging at 12000rpm for 20min to remove impurities, measuring the light absorption value of the supernatant at the wavelength of 216nm, drawing a standard curve by using a gamma-PGA standard substance, and calculating the gamma-PGA content of the bacterial liquid.
As shown in FIG. 8, in the culture medium and the culture conditions, the yield of gamma-PGA of the N14 strain can reach 12.8g/L, and the addition of 1-50 mg/L selenate or 0.1-1 mg/L selenite (measured in Se) has no significant effect on the yield of gamma-PGA.
Example 7 preparation of selenium-enriched Natto by fermenting selenium-enriched Soybean with Bacillus natto N14
1. Production of selenium-rich soybean
The soybean variety is Dongsheng 19, the selenium enrichment of the soybeans is carried out by spraying the biological nano-selenium fermentation liquor in the embodiment 4 on the leaf surfaces in the flowering phase, the application amount is set to be 3, 0.5 g of nano-selenium, 1.2 g of nano-selenium and 4.8g of nano-selenium are respectively applied to each mu, and the control without applying the nano-selenium is set. The soybeans are harvested after being mature, dried in the sun, sorted to remove mildew and crushed seeds, crushed and uniformly mixed, and then the total selenium content is measured by adopting a GB 5009.93 hydride atomic fluorescence spectrometry method. By controlling the application amount of N14 synthesized nano-selenium in soybean flowering period, common soybean and 3 selenium-rich soybeans with different selenium contents are obtained, as shown in figure 9. The content of selenium in soybean of the control group which is not sprayed with nano-selenium is 0.02 +/-0.01 mg/kg, the content of selenium in the selenium-rich soybean 1, the content of selenium-rich soybean 2 and the content of selenium in the selenium-rich soybean 3 which are obtained by using 3 treatments of nano-selenium are respectively 0.64 +/-0.08 mg/kg, 1.37 +/-0.06 mg/kg and 3.49 +/-0.26 mg/kg, and the content of selenium is respectively improved by 32 times, 69 times and 175 times compared with the content of selenium in the control group.
Adopting a shape analyzer (HPLC-HG-AFS) to analyze the selenium shape of the soybeans, accurately weighing 0.500g of selenium-rich soybean 3 samples, placing the samples in a mortar, adding 5mL of protease 14 solution (8 mg/mL), carrying out ultrasonic disruption, placing the mixture at 37 ℃, and carrying out shaking extraction at 150rpm for 12h. Centrifuging at 12000rpm for 10min, collecting supernatant, filtering with 0.22 μm filter membrane, and detecting selenium form on computer, wherein the measurement parameters of form analysis are shown in Table 1. The results showed that selenium-enriched soybeans were dominated by SeMet (96.3%) and contained small amounts of Se (IV) (3.7%). The result shows that the nano selenium obtained by N14 can be absorbed by soybean, and when the total selenium content of the soybean reaches 3.49mg/kg, the content of organic selenium SeMet reaches 96.3%.
2. N14 fermented selenium-rich natto
Selecting plump and mildew-free common and selenium- rich soybeans 1, 2 and 3, cleaning, soaking in deionized water for 12h, draining off water, placing into a culture bottle, sealing with a sealing film, sterilizing with high pressure steam at 121 deg.C for 30 min, and cooling for inoculation. Preparing a seed solution: an N14 single colony is picked and inoculated into an LB test tube, the culture is carried out for 12h at 37 ℃ and 150rpm, 1mL is taken and transferred into a 50mL LB shake flask, and the culture is carried out for 4h at 37 ℃ and 150rpm as seed liquid. Inoculating N14 seed solution according to 6% inoculation amount, fermenting in a 37 deg.C incubator for 24 hr, and aging at 4 deg.C for 24 hr to obtain common natto, selenium-rich natto 1, selenium-rich natto 2 and selenium-rich natto 3. After vacuum freeze drying, crushing by using a mortar, and respectively detecting the total selenium, the selenium form, the nattokinase and the gamma-PGA content.
(1) Content and form of selenium in natto
The total selenium content in the natto is measured by a GB 5009.93 hydride atomic fluorescence spectrometry method. After N14 fermentation, ordinary natto (0.07 +/-0.02 mg/kg) and selenium-rich natto (1-3) with the selenium content of 0.66 +/-0.07, 1.62 +/-0.08 and 3.83 +/-0.22 mg/kg are obtained, natto fermentation of the N14 strain has no obvious influence on the selenium content (figure 9), and the selenium content in the selenium-rich natto is slightly improved compared with soybean.
Analyzing the selenium form of natto by using a form analyzer (HPLC-HG-AFS), accurately weighing 0.500g of selenium-rich natto 3 sample, placing the selenium-rich natto 3 sample in a mortar, adding 5mL of protease 14 solution (8 mg/mL), performing ultrasonic crushing, placing at 37 ℃, and performing vibration extraction at 150rpm for 12h. Centrifuging at 12000rpm for 10min, collecting supernatant, diluting, filtering with 3KD ultrafiltration membrane, and detecting selenium form on computer, wherein the determination parameters of form analysis are shown in Table 1. As shown in fig. 10 (a and B), no Se (IV) was detected in the selenium-rich natto, and the selenium form was 100% of SeMet, indicating that 3.7% of the Se (IV) remaining in the selenium-rich soybean can be completely converted into SeMet by the fermentation process of the N14 selenium-rich natto, and the organic selenium proportion in the selenium-rich natto is 100%.
(2) Influence of N14 selenium-rich natto fermentation on gamma-PGA
Accurately weighing 0.200g natto powder, adding 8mL ultrapure water, uniformly mixing by vortex, carrying out oscillation extraction at 25 ℃ and 160rpm for 1h, taking supernatant, centrifuging at 12000rpm for 20min, sucking 0.3mL supernatant, adding 1.2mL of glacial ethanol, slightly reversing, uniformly mixing, centrifuging at 12000rpm for 10min, discarding supernatant, carrying out vacuum drying on precipitate, redissolving in 1.2mL ultrapure water, properly diluting for 2-5 times, centrifuging at 12000rpm for 20min to remove impurities, detecting OD of supernatant 216 . And drawing a standard curve of the gamma-PGA by adopting the gamma-PGA standard sample, and calculating the content of the gamma-PGA in the sample by utilizing the standard curve. The result shows that the content of the gamma-PGA of the common natto obtained by N14 fermentation is as high as 159.2 +/-17.1 g/kg, while the content of the gamma-PGA of the selenium-enriched natto 1-3 respectively reaches 164.0 +/-14.4 mg/g, 161.0 +/-8.8 mg/g and 158.5 +/-14.0 mg/g (dry weight, figure 11), and the content is equivalent to that of the common natto and has no significant difference. N14 has no obvious influence on the yield of gamma-PGA while obtaining the selenium-rich natto with 100% organic selenium content.
(3) Influence of N14 selenium-rich natto fermentation on nattokinase
The enzymatic activity of the natto kinase is measured by adopting a fibrinolysis-ultraviolet spectrophotometry method of 'DBS 44/013-2019'. Accurately weighing 0.1g natto powder, dissolving with 5mL diluent (containing 2mM calcium sulfate, 10mM sodium chloride, 2mM acetate buffer solution with pH6.0 and 0.005% Triton X-100), ultrasonic extracting for 15min, standing, diluting the supernatant by 5 times, centrifuging at 12000rpm for 20min, and collecting the supernatant for enzyme activity detection.
1.4mL of phosphate buffer (0.01M) and 0.4mL of fibrinogen solution (0.96%) were added to a 10mL centrifuge tube and incubated in a water bath at 37 ℃ for 5min; taking out, adding 0.1mL of thrombin solution, mixing uniformly, and incubating in a water bath at 37 ℃ for 10min; removed, 0.1mL of sample solution added, vortexed for 5s, and incubated in a water bath at 37 ℃ for 60min (vortexed for 5s at 20min and 40min, respectively). At 60min, 2mL of TCA termination reaction solution (0.2M) was added, mixed for 5s, and incubated in a water bath at 37 ℃ for 20min to complete the termination reaction. Centrifuging at 12000rpm for 10min, and collecting the supernatant to determine the absorbance at 275nm (sample solution absorbance A).
Adding TCA into a blank sample tube to terminate the reaction, adding a sample solution, and measuring OD according to the same method 275 (blank tube Absorbance A 0 ). Nattokinase X (FU/g) in the sample is expressed by the formula "X = (A-A) 0 ) and/(0.01X 60X 0.1). Times.D "were calculated. D is the dilution factor of the sample (volume mL/weight g).
The result shows that the Nattokinase (NK) content of the common natto obtained by N14 fermentation is as high as 177.2 +/-14.7 FU/g (dry weight, figure 11), while the NK content of the selenium-enriched natto 1-3 is equivalent to that of the common natto, and has no significant difference. When the selenium-rich natto with 100% organic selenium content is obtained by N14, the enzyme activity of the nattokinase respectively reaches 183.0 +/-22.1 FU/g, 196.9 +/-35.4 FU/g and 174.2 +/-26.2 FU/g (dry weight, figure 11), and the enzyme activity is not reduced.
EXAMPLE 8 Strain N14 tolerance to selenite
1. Strain N14 plate tolerance to selenite on solid plates
Activating freeze-dried Bacillus natto N14 strain on LB plate, selecting single colony, inoculating in LB liquid test tube, performing shake culture at 37 deg.C and 150rpm for 12h, transferring 1mL bacterial liquid into 50mL LB shake flask, performing shake culture at 37 deg.C and 150rpm for 4h, and adjusting bacterial liquid concentration to OD with sterile physiological saline 600 =0.8 as seed liquid. Melting LB solid culture medium, cooling to about 60 deg.C, adding sterile sodium selenite solution (filtering and sterilizing), shaking gently, mixing, and turning to plate to prepare plate containing 0.05-40 mM selenite. Diluting the N14 seed solution with sterile normal saline gradually according to 10 times gradient, respectively adding 2.5 μ L dropwise onto selenium-containing flat plate, drying with sterile air, and culturing at 37 deg.C for 24 hr.
The LB medium formula: 5g/L yeast extract, 10g/L tryptone, 10g/L sodium chloride, pH 7.0-7.2, and 15g/L agar added to solid culture medium.
The tolerance range of the strain N14 to selenite on an LB plate is shown in figure 12, the strain N14 can tolerate selenite up to 20mM, and the colony presents bright red, which indicates that the strain can reduce the selenite into red elemental selenium (biological nano-selenium). The tolerance of the bacillus natto N14 solid plate to selenite is shown in FIG. 12.
2. Strain N14 tolerance to selenite under liquid culture conditions
Activating lyophilized Bacillus natto N14 strain on LB plate, selecting single colony, inoculating in LB liquid test tube, performing shake culture at 37 deg.C and 150rpm for 12 hr, and adjusting bacterial liquid concentration to OD with sterile normal saline 600 =0.8. Preparing NT4 culture medium, sterilizing, adding sterile filtered sodium selenite solution to final concentration of 0.01-100mM, inoculating into the prepared NT4 culture medium according to 1% inoculum size, placing at 37 deg.C, culturing at 150rpm under shaking for 48h, observing thallus growth, and measuring thallus growth by dilution plating method. The results showed that the N14 strain was able to grow at 0.01-100mM, and that N14 was able to tolerate 100mM sodium selenite; the growth of the strain is not significantly influenced in the sodium selenite concentration of 10mM compared with that of a blank control, and the growth of N14 is inhibited by 20.4-85.6% in the sodium selenite concentration of 20-100 mM. The tolerance of Bacillus natto N14 to selenite under liquid culture conditions is shown in FIG. 13.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.
Sequence listing
<110> university of agriculture in China
<120> bacillus subtilis subspecies natto N14 and application thereof
<130> KHP211118127.4
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1433
<212> DNA
<213> Bacillus subtilis subsp. natto
<400> 1
ctatacatgc agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg 60
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taccggatgg ttgtttgaac cgcatggttc aaacataaaa 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 aagtaccgtt 420
cgaatagggc ggtaccttga 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 tgggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct 960
ctgacaatcc tagagatagg acgtcccctt cgggggcaga gtgacaggtg gtgcatggtt 1020
gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc 1080
ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag 1140
gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg 1200
gacagaacaa agggcagcga aaccgcgagg ttaagccaat cccacaaatc tgttctcagt 1260
tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag 1320
catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt 1380
tgtaacaccc gaagtcggtg aggtaacctt taggagccag ccgccgaagt gac 1433
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gcgtctgtcg taaacgcact atcaacagag cttgatgtga cggttcaccg tgacggtaaa 60
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acggatcata caggaacgac gacacatttt gtcccggacc ctgaaatttt ctcagaaaca 180
accgagtatg attatgatct gcttgccaac cgcgtacgtg aattagcctt tttaacaaag 240
ggcgtaaaca tcacgattga ggataaacgt gaaggacaag agcgcaaaaa tgaataccat 300
tacgaaggcg gaattaaaag ttatgtagag tatttaaacc gctctaaaga ggttgtccat 360
gaagagccga tttacattga aggcgaaaag gacggcatta cggttgaagt ggctttgcaa 420
tacaatgaca gctacacaag caacatttac tcgtttacaa acaacattaa cacgtacgaa 480
ggcggtaccc atgaagctgg cttcaaaacg ggcctgactc gtgttatcaa cgattacgcc 540
agaaaaaaag 550
Claims (10)
1. Bacillus subtilis subsp. Natto N14 with preservation number of CGMCC No.21703.
2. A microbial preparation comprising the Bacillus subtilis subspecies natto N14 of claim 1.
3. The method for biosynthesizing nano-selenium by utilizing the bacillus subtilis subspecies natto N14 as described in claim 1, which comprises the following steps: adding selenite with a final concentration of 1-100mM into the fermentation medium, fermenting and culturing the strain N14, and separating and purifying nano selenium from the fermentation product.
4. A method for producing organic selenium by using the bacillus subtilis subsp natto N14 as claimed in claim 1, wherein the method comprises: adding selenite with final concentration of 0.01-0.1mM into fermentation medium, fermenting and culturing strain N14, and separating and purifying organic selenium from fermentation product.
5. The method of claim 4, wherein the organic selenium comprises selenomethylselenocysteine and selenomethionine.
6. The method according to any one of claims 3 to 5, wherein the fermentation medium has a formula of: 18-24g/L glucose, 1.8-2.6g/L glycerol, 4-6g/L yeast extract, 4.2-5.8g/L peptone, 8.2-12.6 g/L-sodium glutamate, 0.2-0.3g/L magnesium sulfate heptahydrate, 2.3-3.6g/L dipotassium hydrogen phosphate and pH 7.0-7.5.
7. The use of the bacillus subtilis subspecies natto N14 of claim 1 for the preparation of selenium-enriched plants.
8. The production method of the selenium-rich soybean is characterized by comprising the following steps:
A. adding selenite with the concentration of 1-100mM into a fermentation culture medium, and fermenting and culturing the bacillus subtilis natto subspecies N14 of claim 1;
B. applying the fermentation liquid obtained in the step A to soil around the rhizosphere of soybean plants or spraying the fermentation liquid to leaf surfaces, and harvesting after the soybeans are mature;
the obtained selenium-rich soybean contains total selenium 0.64-3.49mg/kg and organic selenium 85.0-96.3%.
9. The production method of the selenium-rich natto is characterized by comprising the following steps:
1) Soaking the selenium-rich soybean prepared in claim 8 in water, and steaming the soaked soybean over water;
2) Cooling the cooked soybeans, inoculating the bacillus subtilis subspecies natto N14 as claimed in claim 1, and fermenting and after-ripening to obtain selenium-rich natto;
the obtained selenium-rich natto contains total selenium 0.66-3.83mg/kg, organic selenium 100%, nattokinase 174.2-196.9FU/g, and gamma-polyglutamic acid 158.5-164.0mg/g.
10. The production method of the selenium-rich natto is characterized by comprising the following steps:
(1) Soaking semen glycines in water containing selenite;
(2) Steaming the soaked soybeans in a water-proof way;
(3) Cooling the cooked soybeans, inoculating the bacillus subtilis subspecies natto N14 of claim 1, fermenting and after-ripening to obtain selenium-rich natto;
the obtained selenium-rich natto contains total selenium 0.95-6.42mg/kg, organic selenium 60.5-82.2%, nattokinase 156.4-234.2FU/g, and gamma-polyglutamic acid 149.2-188.0mg/g.
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