CN112481166A

CN112481166A - Method for synthesizing biological nano-selenium by utilizing bacillus methylotrophicus LW-6 and application

Info

Publication number: CN112481166A
Application number: CN202011402939.2A
Authority: CN
Inventors: 唐满仓; 唐少燃; 米哲
Original assignee: Xi'an Hengtian Biotechnology Co ltd
Current assignee: Xi'an Hengtian Biotechnology Co ltd
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2021-03-12
Anticipated expiration: 2040-12-04
Also published as: CN112481166B

Abstract

The embodiment of the specification relates to the technical field of microbiology and biological nano-selenium preparation, in particular to a method for synthesizing biological nano-selenium by utilizing bacillus methylotrophicus LW-6 and application thereof. The method comprises the steps of taking a culture medium containing selenite as a fermentation culture medium, taking bacillus methylotrophicus LW-6 as zymocyte, and fermenting to obtain biological nano-selenium mother liquor containing biological nano-selenium; wherein the preservation number of the Bacillus methylotrophicus LW-6 is CGMCC No. 6793.

Description

Method for synthesizing biological nano-selenium by utilizing bacillus methylotrophicus LW-6 and application

Technical Field

One or more embodiments of the present specification relate to the field of microbiology and biological nano-selenium preparation technology, and in particular, to a method for synthesizing biological nano-selenium by using bacillus methylotrophicus LW-6 and a use thereof.

Background

Selenium is one of the essential trace elements for human, animal and plant, is a component of various enzymes in the body, and plays the roles of resisting diseases, delaying senility, enhancing immunity and the like in life. The organic selenium is used as the active center of glutathione peroxidase, and plays an important role in regulating the antioxidant capacity of animals and plants. 80% of land in China belongs to selenium-deficient land, the content of selenium in the soil is low, and the selenium content of planted agricultural products is also low, so that people in China are in a selenium-deficient state, the best method for solving the problems is to produce selenium-enriched agricultural products, and the problem of producing the selenium-enriched agricultural products is to use selenium-enriched fertilizers and selenium-enriched microbial agents, and biological nano-selenium is a selenium product with the best safety and high bioavailability in organic selenium.

In the early applied selenium supplement preparations, sodium/potassium selenite and sodium/potassium selenate are used as additive components, and the inorganic selenium additive components belong to chemicals with extremely high toxicity, so that the liver is greatly damaged after long-term administration, the original purpose of selenium supplement is contradicted, even the selenium supplement becomes a toxic substitute, and people are no longer enthusiastic to pursue the selenium.

In recent years, along with the great improvement of the cognitive level of medicine and the life of people, the selenium supplement returns to the life theme of people, so that the microbial nano-selenium is generated by transportation. The method greatly avoids and eliminates the adverse effects of high toxicity and easy liver injury of inorganic selenium, is widely applied to the aspects of medical products, health-care food, feed additives, microbial agents and agricultural fertilizers, and obtains remarkable achievement.

Disclosure of Invention

The embodiment of the specification describes a method and application for synthesizing biological nano-selenium by utilizing bacillus methylotrophicus LW-6, and inorganic selenium can be efficiently converted into biological nano-selenium.

In a first aspect, the embodiments of the present specification provide a method for synthesizing biological nano-selenium by using bacillus methylotrophicus LW-6, in which a culture medium containing selenite is used as a fermentation culture medium, bacillus methylotrophicus LW-6 is used as a fermentation bacteria, and fermentation is performed to obtain a biological nano-selenium mother liquor containing biological nano-selenium; wherein the preservation number of the Bacillus methylotrophicus LW-6 is CGMCC No. 6793.

In some embodiments, the concentration of selenite in the fermentation medium is 26mM, and the selenite is sodium selenite.

In some embodiments, the method provided by the first aspect comprises the steps of:

(1) activating Bacillus methylotrophicus LW-6;

(2) inoculating the activated bacillus methylotrophicus LW-6 into a seed culture liquid culture medium to prepare a seed solution;

(3) inoculating the seed liquid into a fermentation culture medium for fermentation; wherein the content of the first and second substances,

in the fermentation process, the fermentation temperature is 25-30 ℃, the stirring speed is 260-280rpm, and the ventilation volume is 1: (0.3-0.6) and the pot pressure is 1.4-1.8F/cm²Fermenting for 48-72 hours to obtain a biological nano selenium mother liquor with the biological nano selenium content of 1800 mg/L;

in the fermentation medium, the initial concentration of peptone is 10g/L, the initial concentration of glucose is 5g/L, KH₂P0₄Has an initial concentration of 2g/L, Mg₂S0₄.7H₂The initial concentration of 0 is lg/L, and the initial concentration of sodium selenite is 4500 mg/L; the initial pH of the fermentation medium was 7.0-7.2 and the initial volume was 70% of the volume of the fermentor.

In some embodiments, the biological nano-selenium mother liquor is a biological nano-selenium zinc mother liquor, and the fermentation medium further comprises zinc sulfate heptahydrate at an initial concentration of 1786 mg/l.

In some embodiments, in step (1), the bacillus methylotrophicus LW-6 is activated using a slant medium; the formula of the slant culture medium is 8g/L glucose, 5g/L yeast extract powder, 10g/L industrial salt, 18-20g/L agar, 10g/L tryptone and water, the balance is supplemented, and the pH is adjusted to 7.0-7.2; during the activation culture period, the culture temperature is 28 ℃, and the culture time is 36 hours;

in the step (2), the activated Bacillus methylotrophicus LW-6 is configured to 10⁸CFU/mL of bacterial suspension; inoculating the bacterial suspension into the seed culture liquid culture medium according to the inoculation amount of 1%;

the formula of the seed culture liquid medium is 8g/L of glucose, 5g/L of yeast extract powder, 10g/L of industrial salt, 10g/L of tryptone and the balance of water; the initial pH of the seed culture liquid medium is 7.0-7.2; during the preparation of the seed solution, the culture temperature is 28 ℃, the shaking culture is carried out at the rotating speed of 150rpm, and the culture time is 24 hours, so as to obtain the seed solution;

in the step (3), the seed solution is inoculated into a fermentation medium in an inoculation amount of 3%.

In a second aspect, the present specification provides a biological nano-selenium mother liquor, which is characterized by being prepared by the method described in the first aspect.

In a third aspect, the embodiment of the specification provides the application of the biological nano selenium mother liquor provided in the second aspect in preparing the selenium-zinc-rich composite microbial fertilizer; wherein, the biological nano selenium mother solution is sprayed on the composite microbial fertilizer particles to prepare the selenium-zinc-rich composite microbial fertilizer.

In a fourth aspect, the embodiment of the specification provides the use of the selenium-zinc-rich composite microbial fertilizer provided by the third aspect in the production of selenium-rich crops; wherein, when the content of the biological nano selenium in the selenium-zinc-rich composite microbial fertilizer is 90 mg/kg;

if the crops are wheat, the dosage of the selenium-zinc-rich compound microbial fertilizer is 40 kg/mu, and the wheat with the selenium content of 279.01 mug/kg can be produced;

if the crops are rice, the dosage of the selenium-zinc-rich compound microbial fertilizer is 40 kg/mu, and the rice with the selenium content of 282.9 mug/kg can be produced;

if the crop is corn, the dosage of the selenium-zinc-rich compound microbial fertilizer is 40 kg/mu, and corn with the selenium content of 217.4 mu g/kg can be produced;

if the crops are soybeans, the dosage of the selenium-zinc-rich compound microbial fertilizer is 40 kg/mu, and soybeans with the selenium content of 218.6 mu g/kg can be produced;

if the crops are peanuts, the dosage of the selenium-zinc-rich composite microbial fertilizer is 40 kg/mu, and the peanuts with selenium content of 197.6 mu g/kg can be produced;

if the crops are millet, the dosage of the selenium-zinc-rich compound microbial fertilizer is 40 kg/mu, and millet with the selenium content of 214.3 mug/kg can be produced;

if the crops are sweet potatoes, the dosage of the selenium-zinc-rich composite microbial fertilizer is 40 kg/mu, and the sweet potatoes with the selenium content of 45.3 mug/kg can be produced;

if the crops are tomatoes, the dosage of the selenium-zinc-rich compound microbial fertilizer is 80 kg/mu, and the tomatoes with the selenium content of 87.9 mug/kg can be produced;

if the crop is eggplant, the dosage of the selenium-zinc-rich composite microbial fertilizer is 80 kg/mu, and the eggplant with the selenium content of 66.1 mug/kg can be produced;

if the crops are cucumbers, the dosage of the selenium-zinc-rich compound microbial fertilizer is 80 kg/mu, and the cucumbers with the selenium content of 42.7 mu g/kg can be produced;

if the crops are apples, the dosage of the selenium-zinc-rich compound microbial fertilizer is 80 kg/mu, and apples with the selenium content of 39.2 mug/kg can be produced;

if the crop is kiwi fruit, the dosage of the selenium-zinc-rich compound microbial fertilizer is 80 kg/mu, and kiwi fruit with the selenium content of 138.8 mug/kg can be produced;

if the crops are tea, the dosage of the selenium-zinc-rich compound microbial fertilizer is 80 kg/mu, and the tea with the selenium content of 3382.7 mug/kg can be produced.

In a fifth aspect, the embodiment of the present specification provides a use of the biological nano-selenium mother liquor provided in the second aspect in preparation of a selenium-zinc-rich microbial agent; adding the biological nano-selenium mother liquor into water, adding auxiliary materials, and stirring to prepare a selenium-zinc-rich microbial agent; the auxiliary material comprises potassium fulvate.

In a sixth aspect, the embodiments of the present specification provide the use of the microbial agent rich in selenium and zinc provided in the fifth aspect in the production of selenium-rich crops; wherein, when the content of the biological nano selenium in the selenium-zinc-rich microbial agent is 90 mg/L;

if the crops are wheat, the dosage of the selenium-zinc-rich microbial agent is 2L/mu, and the wheat with the selenium content of 287.2 mug/kg can be produced;

if the crops are rice, the dosage of the selenium-zinc-rich microbial agent is 2L/mu, and the rice with the selenium content of 214.4 mug/kg can be produced;

if the crop is corn, the dosage of the selenium-zinc-rich microbial agent is 2L/mu, and the corn with the selenium content of 260.1 mug/kg can be produced;

if the crops are soybeans, the dosage of the selenium-zinc-rich microbial agent is 3L/mu, and the soybeans with the selenium content of 237.L mug/kg can be produced;

if the crops are peanuts, the dosage of the selenium-zinc-rich microbial agent is 3L/mu, and the peanuts with the selenium content of 238.L mu g/kg can be produced;

if the crops are millet, the dosage of the selenium-zinc-rich microbial agent is 3L/mu, and millet with the selenium content of 225.5 mug/kg can be produced;

if the crops are sweet potatoes, the dosage of the selenium-zinc-rich microbial agent is 3L/mu, and the sweet potatoes with the selenium content of 60.8 mug/kg can be produced;

if the crops are needle mushrooms, the dosage of the selenium-zinc-rich microbial agent is 1.5L/ton of matrix, and needle mushrooms with the selenium content of 435.5 mug/kg can be produced;

if the crops are the mushrooms, the dosage of the selenium-zinc-rich microbial agent is 1.5L/ton of matrix, and the mushrooms with the selenium content of 1977.4 mug/kg can be produced;

if the crop is agaric, the dosage of the selenium-zinc-rich microbial agent is 1.5L/ton of matrix, and agaric with the selenium content of 2853.2 mug/kg can be produced;

if the crops are tomatoes, the dosage of the selenium-zinc-rich microbial agent is 4L/mu, and the tomatoes with the selenium content of 97.9 mug/kg can be produced;

if the crops are eggplants, the dosage of the selenium-zinc-rich microbial agent is 4L/mu, and the eggplants with the selenium content of 76.2 mug/kg can be produced;

if the crops are cucumbers, the dosage of the selenium-zinc-rich microbial agent is 4L/mu, and the cucumbers with the selenium content of 49.7 mug/kg can be produced;

if the crops are apples, the dosage of the selenium-zinc-rich microbial agent is 5L/mu, and the apples with the selenium content of 37.3 mug/kg can be produced;

if the crop is kiwi fruit, the dosage of the selenium-zinc-rich microbial agent is 5L/mu, and kiwi fruit with the selenium content of 168.8 mug/kg can be produced;

if the crops are tea, the dosage of the selenium-zinc-rich microbial agent is 4L/mu, and the tea with the selenium content of 3438.6 mug/kg can be produced.

In the embodiment of the specification, the biological nano selenium and zinc is synthesized by utilizing the bacillus methylotrophicus LW-6, and the conversion rate of selenium is improved by 15-20% compared with that of other strains such as bacillus subtilis and the like; the prepared biological nano-selenium-zinc can be applied to selenium-zinc-rich fertilizers; the biological nano-selenium-zinc fertilizer has the characteristics of environmental friendliness, high yield, safety, high efficiency and the like, and the biological nano-selenium-zinc fertilizer obtained by production is used for a selenium-zinc-rich fertilizer, and after the fertilizer is applied, the effects of crops, melons, fruits, vegetables and the selenium-zinc-rich fertilizer are obvious.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 shows a molecular evolutionary tree of Bacillus methylotrophicus LW-6 provided in the examples of the present specification.

FIG. 2 is a graph showing the tolerance of Bacillus methylotrophicus LW-6 to various sodium selenite concentrations as provided in the examples herein.

FIG. 3A is a graph showing the biological nanoselenium production of Bacillus methylotrophicus LW-6 at different sodium selenite concentrations as provided in the examples herein;

FIG. 3B is a graph showing biological nanoselenium conversion of Bacillus methylotrophicus LW-6 at different sodium selenite concentrations as provided in the examples herein;

FIG. 4 is a Transmission Electron Microscope (TEM) photograph of purified nano-selenium particles transformed by Bacillus methylotrophicus LW-6 provided in the examples herein.

Detailed Description

The biological nano-selenium as the plant health activator has the characteristics of improving the activity of biological enzymes in plants, increasing the stress resistance of crops, promoting the healthy growth of plants and the like. Is suitable for the selenium enrichment of various crops such as grains, vegetables, tea leaves, fruits and the like. The selenium can prevent crops from absorbing heavy metals and promote the discharge of heavy metals in human bodies and animal bodies, the former can be used for soil seriously polluted by the heavy metals, and the latter can reduce the accumulation of the heavy metals in the human bodies and the animal bodies.

Biological nano selenium constructs genetic engineering bacteria through a synthetic microorganism technology, promotes mass propagation of the engineering bacteria under a proper condition, then related substrates such as sodium selenite and the like are added, and the biological fermentation technology and the biological organic selenium are fused through the nanotechnology through fermentation of microorganisms to form the biological organic nano selenium which can be efficiently utilized by plants, so that the major problem that selenium cannot participate in plant growth for a long time is solved, the toxicity of the biological organic nano selenium is one twentieth of that of inorganic selenium, and the product efficiency is greatly improved. Has important effects on promoting the quality safety of agricultural products, reducing the using amount of agricultural products to pesticides and fertilizers, improving the quality of the agricultural products and developing ecological agriculture healthily. The product can obviously promote the yield increase of crops after being used, and can greatly improve the quality and the flavor of the crops. Such as waxy nature of the grain, sweetness of the fruit, and mouthfeel. The nano selenium is adsorbed on natural polysaccharide with biocompatibility and applied to rice planting, the rice yield is improved by 8-15%, the selenium content is improved by 3-10 times, the total antioxidant capacity of nutrient substances in rice is improved by 30-60%, the nano selenium is used in the tea planting process, the tea antioxidant property can be improved, the bitter components of the tea can be reduced, the nano selenium is used in the planting process of the chickpea, the fruit maturity can be obviously improved, the color can be changed in advance, and the selenium content can reach the standard of selenium-enriched fruits through detection. The nano-selenium has the unique biological effects of low toxicity, high absorption and utilization rate, high efficiency, oxidation resistance and the like, can improve the yield of agricultural products, enhance the oxidation resistance of plants, improve the quality and the flavor of the agricultural products and greatly reduce the use of pesticides. The yield increasing effect is obvious, and the fruit quality is obviously improved. Experiments show that: after the biological nano selenium product is applied to tomatoes, leaves are taller, thicker and green in the plant growth process, and the growth vigor is better. The color of the tomato is changed earlier, the fruit type is better, the size is more uniform, and the ripe tomato has rich fragrance and good taste. The selenium content of the harvested tomatoes is improved by over 0.03 percent through detection, so that the small tomatoes can be in a selenium-rich state from zero. After the nano-selenium is applied to the leaf surfaces of the celery, the contents of indexes such as celery chlorophyll, soluble sugar, protein, carotene and the like are respectively increased by 26.1%, 70.4%, 37.1% and 61.4%.

Zinc is one of the essential trace elements for plant and human body, and plays an important role in the development process of plant and human body. Is usually praised as 'flower of life' and 'source of intelligence', and human zinc deficiency can reduce human immunity, influence normal metabolism and cause various diseases. The zinc deficiency of plants can also cause various physiological diseases, and the improvement of the yield and the quality of crops is seriously influenced. At present, inorganic zinc is mainly used for supplementing zinc for plants and human bodies, the absorption utilization rate of the inorganic zinc is low, antagonism is easy to occur with cations such as calcium, magnesium and the like, and particularly in the transportation and conduction process of the plants in the bodies, the inorganic zinc is easy to react with anions to form insoluble substances, so that the absorption efficiency is reduced. The zinc element of the biological nano-selenium-zinc synthesized by microbial conversion and amino acid and other small molecules form stable chelate. Thereby making plants and human bodies more easily absorbed. The utilization rate of the zinc element is improved.

The specification provides a method for biologically synthesizing nano selenium and zinc by utilizing bacillus methylotrophicus LW-6 and application thereof.

Before expanding on the solution provided by the illustrative embodiment, the following description is provided.

It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989 and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1 isolation and identification of bacterial species

The inventor of the application separates and purifies from soil, and obtains a Bacillus methylotrophicus LW-6(Bacillus methylotrophicus) strain which can tolerate sodium selenite with higher concentration through the improvement of a gene mutagenesis technology, wherein the Bacillus methylotrophicus LW-6 is stored in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the address is the great Tutunning road of the south Kogyo of Beijing, the institute of microorganisms of China academy of sciences, the postal code is 100101, the storage number is CGMCC No.6793, and the storage date is 11 months and 7 days in 2012.

The identification process is as follows.

1.1, amplifying 16S rDNA and gyrA gene sequences by PCR and sequencing:

inoculating the strain LW-6 into LB solid medium for culturing for 24h, taking 0.2mL of a sterilized PCR tube, and adding 10 muL of ddH₂And O, picking a single colony from the aseptic toothpick, and uniformly stirring and mixing the single colony in a PCR tube.

1.2, constructing a PCR reaction system:

16S rDNA amplification:

as set forth in SEQ ID NO: 1 (specific sequence: 5'-cgggatccagagtttgatcctggctcagaacgaacgct-3') and SEQ ID NO: 1506R (specific sequence: 5'-cgggatcctacggctaccttgttacgacttcacccc-3') shown in 2 is used as a primer, and a 16S rDNA gene sequence is obtained by PCR amplification. The PCR reaction system is as follows: ddH ₂0, 18.5 μ L; 10 xBuffer, 2.5 uL, dNTP Mix, 2 uL; 8F, 0.5 μ L; 1506R, 0.5 μ L; 0.5 mu L of bacterial liquid; rTaqDNA polymerase, 0.5. mu.L.

The PCR reaction conditions are as follows: l0min at 94 ℃; 30 cycles of 94 ℃, 40s, 56 ℃, 40s, 72 ℃ and 1 min; 72 ℃ for 10 min; storing at 4 ℃.

gyrA amplification; as set forth in SEQ ID NO: p is shown in 3

gyrA

F (concrete sequence: 5'

cagtcaggaaatgcgtacgtcctt

3') and SEQ ID NO: p is shown as 4

gyrA

R (concrete sequence: 5'

caaggtaatgctccaggcattgct-3') as primers, and obtaining the gyrA gene sequence by PCR amplification. The PCR reaction system is as follows: ddH₂O，18.5μL；10×Buffer，2.5μL；dNTP Mix，2μL；p

gyrA

F，0.5μL；p

gyrA

R, 0.5 μ L; 0.5 mu L of bacterial liquid; rTaqDNA polymerase, 0.5. mu.L.

The PCR reaction conditions are 95 ℃ and 5 min; 94 ℃ for 1 min; 55

At 62 ℃ for 1 min; 72 ℃ for 2 min; a total of 30 cycles; 72 ℃ for 10 min; storing at 4 ℃.

And purifying and sequencing the DNA fragment obtained by PCR amplification, and splicing the sequencing result by using DNAMAN software. The determined sequence of 16S rDNA (SEQ ID NO: 5, specifically actccgtcggacttcagagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtttgaaccgcatggttcagacataaaaggtggcttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaagaacaagtgccgttcaaatagggcggcaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggcgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaag6cgactctctggtctgtaactgacgct aggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccga gtcggttgaggtaaccttttaggccagccagccagccagccagccaggcaagtgggatgtgatgttgatggtgagtcg) and the sequence of gyrA gene (SEQ ID N0: 6, specifically acagtccgccagtttgcccggcagattggaaatctcaagcgcacttttgcggcgggtcaattcccgcgcttttttcgctgccatccgcgctcttgcggccattaaacctttttcaacgattttgcgggctgagtccggattttcaagaaggaatgtttccagcgcagaagaaaacagcgtatcagtgatcgttctcgcttcggagttgccgagcttcgttttcgtctgcccttcgaattgcggatcagggtgcttaattgaaataatggcagtcagcccttccctcacatcatccccgcttaaattcggatcattttctttgaaaatcccttttcttcttgcatagtcgtttataacacgggtcagaccggttttaaatccggcttcgtgcgt ccgccctttcggtatgtatgtgttggttgaaatagtaaatagtgcttgtcgttgtattgcaatgacttcaacttcaacttcaacttcaacgttacgttactgccttgaggcttgcgtgcgtgcgtgcgtgcgtgcttgcttgctaggcttgcttaaggcttgcttgcgtgcttgcatcgtgcactgcatgcatgcttgcttgctacaggcttgcttgcttgcgtgcttgcgtgcttgcttgcttgccgttgctcgtgcttgcttgcttgctcgtgcttgccgtcgcttgcttgcttgcttgcttgctcgcttgcatcgtgcatcgtgcatcgatgcttgcttgcatcgtgcaggcaggcttgcttgcttgcgcgcgcttgcaggccgttgctacatgcttgcgcttgcgcgcgcgcttgcttgcttgcgcgcgcttgcttgcttgcttgcttgcttgcttgcgcgcttgcttgcaggcttgcttgcttgcgcttgcaggcaggcaggcttgcgcaggcttgcttgcgcgcgcgcttgctacatgcttgcttgcttgcttgcttgcttgcaggcttgcttgcaggcttgcttgcttgcttgcttgcttgcttgcttgcaggctacatgctacatgcaggcttgcttgctacatgcttgcttgcgctacatgcttgcaggctacatgcttgcttgcttgcttgcttgctacatgcttgcttgcttgcttgcttgcttgcttgcttgcaattgcttgcttgctacatgcttgctacatgcttgcttgcttgctacatgcttgcttgcttgcttgcttgccaaggcttgcttgcttgcttgcttgcgcgcttgcttgcttgcttgcttgcttgcttgcttgcttgctacatgcttgcttgctacatgcttgcttgcttgc. Therefore, the LW-6 strain is determined to be Bacillus methylotrophicus, named as Bacillus methylotrophicus LW-6(Bacillus methylotrophicus), and is stored in China general microbiological culture collection center with the strain preservation number of CGMCC No. 6793.

Example 2 tolerant concentration of sodium selenite by Bacillus methylotrophicus LW-6.

2.1, preparing solid LB selenium-containing culture medium with different concentrations (each liter of culture medium contains 10g of NaCl, 10g of pancreatic gland, 5g of yeast extract, 15g of agar and 1L of deionized water), and autoclaving at 121 ℃ for 20 min. Preparing 1M sodium selenite mother liquor, filtering, sterilizing, adding sodium selenite solution to make the sodium selenite content in the culture medium respectively 0mM, 10mM, 25mM, 35mM, 55mM, 70mM and 80 mM.

2.2, a single colony of the Bacillus methylotrophicus LW-6 strain was inoculated into 5ml of LB liquid medium and shaken for 8 hours (150rpm, 28 ℃). Taking the bacteria liquid obtained by shaking culture, and diluting the bacteria liquid into mother liquid with OD600 equal to 0.8 for later use; respectively diluting the mother liquor to 10^-2、10^-3、10^-4、10^-5、10^-6Separately, 2.5. mu.L of bacterial solutions of different concentrations were dropped onto the selenium-containing plates, and 6 replicates of each concentration were cultured at 28 ℃ for 48 hours, and the growth and color change of colonies were observed, and the results are shown in FIG. 2. Wherein, if the bacterial growth is inhibited, the bacterial colony is sparse and the color is light.

From the results, it can be seen that 10mM, 25mM and 35mM of sodium selenite had no significant inhibitory effect on the growth of Bacillus methylotrophicus LW-6; 55mM and 70mM sodium selenite have obvious inhibition effect on the Bacillus methylotrophicus LW-6; in the presence of 80mM sodium selenite, the Bacillus methylotrophicus LW-6 did not grow, thereby obtaining the Bacillus methylotrophicus LW-6 with a sodium selenite tolerance concentration of 70mM, i.e., 12.11 g/L.

Example 3 transformation efficiency of Bacillus methylotrophicus LW-6 for sodium selenite.

3.1, preparing liquid LB culture medium containing selenium with different concentrations, taking 5mL of the liquid LB culture medium, subpackaging the liquid LB culture medium in test tubes, and autoclaving at 121 ℃ for 20 min. Preparing 1M sodium selenite solution, filtering and sterilizing. Different amounts of sodium selenite solution were added to LB medium to achieve culture sodium selenite content of 0mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, respectively, with 3 replicates per concentration gradient.

3.2, a single colony of the Bacillus methylotrophicus LW-6 strain was inoculated into 5ml of LB liquid medium and shaken for 8h (150rpm, 28 ℃). Diluting the cultured bacterial liquid to OD₆₀₀0.8. The diluted bacterial solution was inoculated into LB medium (containing sodium selenite) at an inoculum size of 0.1% and cultured with shaking for 48 hours.

There are two ways to determine the content of bio-nano-selenium in the bacterial solution obtained by 48 hours of shake culture in step 3.2.

Mode 1 preparation of 1M Na with distilled Water₂S solution (ready for use); collecting 1ml of culture solution containing no selenite and different amount of nano-selenium suspension, centrifuging at 12000r/min for 5min in 1.5ml centrifuge tube, removing supernatant, cleaning for 3 times, and adding 1ml of 1M Na₂The S solution is mixed evenly and then fully reacted for l hour, and then is centrifuged for 2min at 12000 r/min; the supernatant was then taken and absorbance measured at 500 nm. And according to the measurement result, a nano-selenium absorbance standard curve is prepared. And then, measuring the absorbance of the bacterial liquid obtained by shaking culture for 48 hours in the step 3.2, and obtaining the biological nano-selenium content in each sample by using a nano-selenium absorbance standard curve. Where each sample had three replicates, i.e. 3 determinations per sample.

And 2, measuring the amount of selenious acid ions in the bacterial liquid obtained by shaking culture for 48 hours in the step 3.2, namely measuring the content of the residual inorganic selenium after fermentation. Specifically, the content of inorganic selenium can be determined by the method described in national standard DBS 42/010-. Then, according to the content of the residual inorganic selenium and the adding amount of the inorganic selenium, the amount of the biological nano selenium converted by the microorganism can be calculated.

Fig. 3A shows the yield of biological nano-selenium at different sodium selenite concentrations.

Fig. 3B shows the conversion rate of bio-nano-selenium at different concentrations of sodium selenite, wherein the conversion rate is calculated by measuring the amount of bio-nano-selenium by means of the above-mentioned mode 2.

As can be seen from FIGS. 3A and 3B, the Bacillus methylotrophicus LW-6 strain can convert sodium selenite to biological nano-selenium to a greater extent at a lower concentration. The biological nano selenium obtained by conversion is nano-sized simple substance selenium formed by combining organic substances such as micromolecular peptides and the like in the microbial metabolism process, is more beneficial to plant absorption and is safer. From the yields and the conversion rates shown in FIGS. 3A and 3B, it can be deduced that the yield of the nano-selenium synthesized by Bacillus methylotrophicus LW-6 reaches the highest value of 1800mg/L and the conversion rate of sodium selenite reaches 88.3% when the content of sodium selenite in the liquid culture medium is 26 mM.

Example 4 biological Nanoselenium profiling

4.1, activating Bacillus methylotrophicus LW-6, transferring 0.1% bacterial liquid (OD600 ═ 0.8) into a sterilized conical flask containing LB liquid culture medium, adding 1M sodium selenite mother liquor to make the final concentration of sodium selenite be 5mM, placing in a shaking table, 28 ℃,150rpm, and culturing for 48 h.

4.2, taking out the red bacterial liquid after 48 hours of shake culture, centrifuging for 10 minutes at 4000r/min at normal temperature, removing the supernatant, resuspending and precipitating with normal saline, centrifugally washing for 3 times, taking about 20 mu L of red mixed liquid of the bacteria and the nano selenium, dripping the red mixed liquid on a carbon-supported membrane copper net, absorbing excessive moisture by using filter paper, airing, observing under a transmission electron microscope (TEM, JEM-1230Japan), and analyzing the biological nano particles by using an energy spectrum analyzer (EDX).

As shown in FIG. 4, spherical nano-selenium particles with a particle size of about 150nm were observed on the cell membrane of Bacillus methylotrophicus LW-6 under a transmission electron microscope. Analysis of nanoparticles indicated by red arrows by EDX spectroscopy indicated that specific absorption peaks of selenium appeared at 1.37, 11.22 and 12.49keV, respectively, indicating that the nanoparticles formed after transformation of sodium selenite by Bacillus methylotrophicus LW-6 strain were nano-selenium.

Example 5 fermentation Process of biological Nano selenium Zinc

5.1, activation of the Strain

Performing activation culture on the strain by using a slant culture medium, wherein the formula of the culture medium comprises 8g/L of glucose, 5g/L of yeast extract powder, 10g/L of industrial salt, 18-20g/L of agar, 10g/L of tryptone, and water which is supplemented to 1L, and adjusting the pH to 7.0-7.2; bacillus methylotrophicus LW-6 was inoculated on a slant medium and cultured at 28 ℃ for 36 hours.

5.2 preparation of seed solutions

The formula of the seed culture liquid culture medium is as follows: 8g/L of glucose, 5g/L of yeast extract powder, 10g/L of industrial salt, 10g/L of tryptone and water, wherein the pH value is adjusted to 7.0-7.2. Preparing the activated bacillus methylotrophicus LW-6 into 10 by using sterile normal saline⁸The bacterial suspension of CFU/mL is inoculated in a liquid culture medium by the inoculation amount of 1 percent, and is subjected to shaking culture at the temperature of 28 ℃ by a shaking table, the rotating speed is 150rpm, and the culture time is 24 hours.

5.3, fermentation in fermenter

The fermentation medium comprises 10g/L protein, 5g/L glucose and KH₂P04 2g/L，Mg₂S0₄.7H₂0 lg/L, 4500 mg/L sodium selenite, 1786 mg/L zinc sulfate heptahydrate, and pH 7.0-7.2; controlling the volume of the culture medium to be 70% of the volume of the fermentation tank, inoculating the seed liquid into the fermentation tank according to the inoculation amount of 3%, controlling the fermentation temperature to be 28 ℃, the stirring speed to be 280rpm, and the ventilation volume to be 1: (0.3-0.6), the pot pressure is 1.4-1.8F/cm²And fermenting for 48-72 hours.

5.4, obtaining 1800mg/L biological nano-selenium-zinc mother liquor by fermentation liquor.

And (4) putting the fermentation liquor into a tank, and measuring the content of the biological nano selenium to obtain a biological nano selenium mother liquor with the content of the biological nano selenium of 1800 mg/L. Wherein, zinc is not converted by microorganism, and the state of inorganic zinc at the initial addition is considered in the biological nano-selenium mother liquor. Wherein, the biological nano selenium mother liquor can also be called biological nano selenium zinc mother liquor.

Example 6 application of biological Nano selenium mother liquor in liquid Fertilizer

Adding the biological nano selenium zinc mother liquor with the biological nano selenium content of 1800mg/L into purified water in a stirring kettle, adding auxiliary materials such as potassium fulvate and the like, and fully and uniformly stirring to prepare the selenium-zinc-rich microbial agent with the selenium content of 90mg/L and the zinc content of 20 mg/L.

The selenium-rich microbial agent is applied to grain crops such as selenium-rich wheat, rice, corn and the like, coarse cereals such as selenium-rich soybean, peanut, millet and sweet potato and the like in edible fungi such as selenium-rich needle mushroom, agaric and the like, the dosage of fruits such as selenium-rich apple, kiwi fruit and the like and selenium-rich tea are respectively 2L/mu, 3L/mu, 1.5L/ton matrix, 4L/mu, 5L/mu and 4L/mu, the selenium content of each crop is respectively 287.2 mu g/kg, rice 214.4 mu g/kg, corn 260.1 mu g/kg, soybean 237.L mu g/kg, peanut 238. mu g/kg, millet 225.5 mu g/kg, sweet potato 60.8 mu g/kg, needle mushroom 435.5 mu g/kg, mushroom 1977.4 mu g/kg, agaric 2853.2 mu g/kg, tomato 97.9 mu g/kg, 76.2 mug/kg eggplant, 49.7 mug/kg cucumber, 37.3 mug/kg apple, 168.8 mug/kg kiwi and 3438.6 mug/kg tea.

Example 7 application of biological Nano selenium mother liquor in solid Fertilizer

Spraying the biological nano selenium-zinc mother liquor with the biological nano selenium content of 1800mg/L onto the composite microbial fertilizer particles in a mixer to process the selenium-zinc-rich composite microbial fertilizer, wherein the nano selenium content is 90mg/kg, and the zinc content is 20 mg/kg.

The selenium-rich composite microbial fertilizer is applied to grain crops such as selenium-rich wheat, rice, corn and the like, coarse cereals such as selenium-rich soybean, peanut, millet and sweet potato and the like, cucumbers such as selenium-rich tomato, eggplant, cucumber and the like, fruits such as selenium-rich apple and kiwi fruit and the like, wherein the selenium content of each crop of the selenium-rich tea is respectively 40 kg/mu, 80 kg/mu and 80 kg/mu, and the selenium content of each crop of the selenium-rich tea is respectively 279.01 mu g/kg of wheat, 282.9 mu g/kg of rice, 217.4 mu g/kg of corn, 218.6 mu g/kg of soybean, 197.6 mu g/kg of peanut, 214.3 mu g/kg of millet, 45.3 mu g/kg of sweet potato, 87.9 mu g/kg of tomato, 66.l mu g/kg of eggplant, 42.7 mu g/kg of cucumber, 39.2 mu g/kg of apple, 138.8 mu g/kg of kiwi fruit and 3382.7 mu g.

The embodiment of the specification utilizes bacillus methylotrophicus LW-6 to synthesize biological nano-selenium and zinc, and is applied to a selenium and zinc rich fertilizer. The biological nano selenium-zinc is prepared by a biological fermentation process, and has the characteristics of environmental friendliness, high yield, safety, high efficiency and the like, and the biological nano selenium-zinc obtained by production is used for a selenium-zinc-rich fertilizer, and after fertilization, the effects of crops, melons, fruits, vegetables and the selenium-zinc-rich fertilizer are obvious.

In the embodiment of the specification, the biological nano selenium and zinc is synthesized by utilizing the bacillus methylotrophicus LW-6, and the conversion rate of selenium is improved by 15-20% compared with that of other strains such as bacillus subtilis and the like.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Sequence listing

<110> Xian Hengtian Biotechnology Ltd

<120> method for synthesizing biological nano-selenium by utilizing bacillus methylotrophicus LW-6 and application

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cgggatccag agtttgatcc tggctcagaa cgaacgct 38

<210> 2

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgggatccta cggctacctt gttacgactt cacccc 36

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cagtcaggaa atgcgtacgt cctt 24

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

caaggtaatg ctccaggcat tgct 24

<210> 5

<211> 1455

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

actccgtcgg acttcagagc ggacagatgg gagcttgctc cctgatgtta gcggcggacg 60

ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga aaccggggct 120

aataccggat ggttgtttga accgcatggt tcagacataa aaggtggctt cggctaccac 180

ttacagatgg acccgcggcg cattagctag ttggtgaggt aacggctcac caaggcgacg 240

atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc 300

ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc 360

gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg ttagggaaga acaagtgccg 420

ttcaaatagg gcggcacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca 480

gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagggctc 540

gcaggcggtt tcttaagtct gatgtgaaag cccccggctc aaccggggag ggtcattgga 600

aactggggaa cttgagtgca gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg 660

tagagatgtg gaggaacacc agtggcgaag cgactctctg gtctgtaact gacgctagga 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 agtcggtgag gtaacctttt aggagccagc cgccgaaggt gggacagatg 1440

attggggtga agtcg 1455

<210> 6

<211> 867

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

acagtccgcc agtttgcccg gcagattgga aatctcaagc gcacttttgc ggcgggtcaa 60

ttcccgcgct tttttcgctg ccatccgcgc tcttgcggcc attaaacctt tttcaacgat 120

tttgcgggct gagtccggat tttcaagaag gaatgtttcc agcgcagaag aaaacagcgt 180

atcagtgatc gttctcgctt cggagttgcc gagcttcgtt ttcgtctgcc cttcgaattg 240

cggatcaggg tgcttaattg aaataatggc agtcagccct tccctcacat catccccgct 300

taaattcgga tcattttctt tgaaaatccc ttttcttctt gcatagtcgt ttataacacg 360

ggtcagaccg gttttaaatc cggcttcgtg cgtccgcctt cgtatgtgtt gatattattt 420

gtgaaagaat aaatattgct tgtatagctg tcgttgtatt gcaatgcaac ttcaaccgtt 480

atgccgtctt tctcgccttc gatataaatc ggctcttcat gaacgacttc tttggaacgg 540

tttaagtact caacatagct tttgattccg ccttcgtagt ggtactcgtt tttccgttct 600

tgtccttcac gtttgtcttc aatcgtgatg tttacacctt ttgtcaggaa ggccaattcc 660

cggacacggt ttgaaagcag gtcatagtcg tattcggttg tttctttgaa aatttccgga 720

tccggaacga agtgcgtaat cgttccggtc ttatcagtat caccgatcac ttcaagatcg 780

gccacaggta caccgcgctc gtacgcctga tagtgggatt tttccgtcac gatgaaccgt 840

aacgtcaaga gtggtcgaca aggcgtt 867

Claims

1. A method for synthesizing biological nano-selenium by utilizing Bacillus methylotrophicus LW-6 is characterized in that a culture medium containing selenite is taken as a fermentation culture medium, the Bacillus methylotrophicus LW-6 is taken as zymocyte, and fermentation is carried out to obtain biological nano-selenium mother liquor containing the biological nano-selenium; wherein the preservation number of the Bacillus methylotrophicus LW-6 is CGMCC No. 6793.

2. The method according to claim 1, wherein the selenite is sodium selenite and the concentration of selenite is 26mM in the fermentation medium.

3. The method of claim 1, comprising the steps of:

(1) activating Bacillus methylotrophicus LW-6;

4. The method of claim 3, wherein the biological nano-selenium mother liquor is a biological nano-selenium zinc mother liquor, and the fermentation medium further comprises zinc sulfate heptahydrate at an initial concentration of 1786 mg/L.

5. The method according to claim 3, wherein in the step (1), the Bacillus methylotrophicus LW-6 is activated using a slant medium; the formula of the slant culture medium is 8g/L glucose, 5g/L yeast extract powder, 10g/L industrial salt, 18-20g/L agar, 10g/L tryptone and water, the balance is supplemented, and the pH is adjusted to 7.0-7.2; during the activation culture period, the culture temperature is 28 ℃, and the culture time is 36 hours;

6. A biological nano-selenium mother liquor, which is prepared by the method of any one of claims 1 to 5.

7. Use of the biological nano-selenium mother liquor of claim 6 in preparation of a selenium-zinc-rich composite microbial fertilizer; wherein, the biological nano selenium mother solution is sprayed on the composite microbial fertilizer particles to prepare the selenium-zinc-rich composite microbial fertilizer.

8. Use of the selenium and zinc rich composite microbial fertilizer of claim 7 in the production of selenium rich crops; wherein, when the content of the biological nano selenium in the selenium-zinc-rich composite microbial fertilizer is 90 mg/kg;

9. The use of the biological nano-selenium mother liquor of claim 6 in the preparation of a selenium and zinc-rich microbial agent; adding the biological nano-selenium mother liquor into water, adding auxiliary materials, and stirring to prepare a selenium-zinc-rich microbial agent; the auxiliary material comprises potassium fulvate.

10. Use of the selenium and zinc enriched microbial inoculant of claim 9 in the production of selenium enriched crops; wherein, when the content of the biological nano selenium in the selenium-zinc-rich microbial agent is 90 mg/L;