An isolated Aeromonas caviae and its application in the preparation of nano-selenium

Abstract

The present invention relates to the field of biotechnology, and discloses a separated Aeromonas caviae and its application in the preparation of nano-selenium. The preservation number of the Aeromonas caviae is CCTCC NO: M2025128. The present invention for the first time provides an Aeromonas caviae with both selenium tolerance and selenium reduction ability, which can tolerate high-concentration selenite culture solution and synthesize biological nano-selenium. The nano-selenium nutrient solution can be prepared by the Aeromonas caviae Se01 provided by the present invention and applied to selenium-rich agricultural planting, and has a significant selenium biofortification effect on rape, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the fields of microbial application and biosynthesis of nano-selenium. Specifically, it relates to an isolated Aeromonas caviae and its application in the preparation of nano-selenium. The applicant obtained a strain that can efficiently reduce selenite and synthesize biological nano-selenium through the isolation, purification, and identification of the strains adsorbed on the roots of rape plants. Background Art

[0002] Selenium, as an important trace element essential for humans and animals, is closely related to the health of the body. Selenium deficiency can cause Keshan disease, liver cirrhosis, asthma, cardiovascular diseases, and cancer, etc. Appropriate selenium supplementation can prevent the occurrence of chronic diseases and is beneficial to the nervous system, immune system, and reproductive system. The WHO recommends that the daily selenium supplementation for adults should be 40 - 400 μg. Since the safety usage threshold of inorganic selenium is relatively narrow compared to the harmful threshold to the human body, using the organic selenium already transformed in plants as a dietary selenium source to meet the daily selenium supplementation needs is currently recognized as the safest and most effective selenium supplementation method.

[0003] At present, the selenium content in crops is mainly improved by foliar spraying or soil application of selenium fertilizers. Sodium selenite is the main selenium fertilizer, but different crops have significant differences in their absorption and conversion abilities, and frequent application is often required to achieve an increase in selenium nutritional level. The application of inorganic selenium fertilizers in agricultural practices results in high production costs and causes selenium pollution in the soil and rivers to varying degrees. With the development of technology, biological nano-selenium has attracted much attention. The main reasons are that nano-selenium has a high surface area - volume ratio and is more easily absorbed by plants; compared with nano-selenium synthesized by traditional physical or chemical means, biological nano-selenium has a more regular shape, and the amino acid modification on its surface endows it with high biological activity and stability; in addition, biological nano-selenium has a high adhesion ability and does not require multiple applications, which is safer and more effective than inorganic selenium fertilizers. Therefore, biological nano-selenium is in line with the sustainable development of agriculture and has broad application prospects in future selenium-rich agriculture and environmental governance, etc.

[0004] As a key raw material for the synthesis of biological nano-selenium, inorganic selenium usually has high cytotoxicity, inhibits the growth of many microorganisms and even causes their death; some strains with selenium tolerance lack the ability to convert selenium and are difficult to synthesize nano-selenium. Therefore, it is of great significance to explore microbial strains with both selenium tolerance and selenium reduction ability.

[0005] Rapeseed, a Brassica crop, has the ability to efficiently absorb, transform, and enrich selenium, and is regarded as an excellent bioreactor for selenium, attracting much attention among selenium-enriched vegetables. The applicant isolated a strain of Aeromonas caviae with both selenium tolerance and selenium reduction ability from the roots of rapeseed grown in a hydroponic nutrient solution containing 40 μM sodium selenite. Through liquid culture, inorganic selenium can be converted into biological nano-selenium. The prepared biological nano-selenium can be applied to the selenium-enriched planting process of rapeseed, achieving good results and having broad application prospects. Summary of the Invention

[0006] The object of the present invention is to provide an isolated strain of Aeromonas caviae ( Aeromonas caviae ), which can efficiently synthesize biological nano-selenium and be applied to production practice. The preservation number of the Aeromonas caviae is CCTCC NO: M2025128.

[0007] Another object of the present invention is to provide a method for preparing biological nano-selenium, which is simple, efficient, and convenient, and suitable for large-scale promotion.

[0008] The last object of the present invention is to provide the application of Aeromonas caviae ( Aeromonas caviae ), and the strain can reduce or transform selenium elements under high inorganic selenium concentration.

[0009] To achieve the above objects, the present invention takes the following technical measures:

[0010] The applicant collected the roots of rapeseed grown in a hydroponic nutrient solution containing 40 μM sodium selenite at the Oil Crops Research Institute, Chinese Academy of Agricultural Sciences. After being treated with an ultrasonic crusher, it was added to an LB medium for enrichment culture. A certain amount of the culture solution was spread on a separation plate (LB solid medium containing 2 mmol / L sodium selenite), and after culturing for 48 h, red colonies were picked. After several times of separation and purification, several clones were obtained. The separated strains were subjected to Gram staining and microscopic examination to observe their bacterial morphological characteristics. Amplification was carried out using the universal primers for bacterial identification 16S rDNA (the primer pair sequences are: forward F primer: 5’-AGTTTGATCMTGGCTCAG-3’; reverse R primer: 5’-GGTTACCTTGTTACGACTT-3’), and after sequencing and comparison, bacterial identification was carried out. Among them, the Se01 strain was identified as Aeromonas caviae ( Aeromonas caviae ).

[0011] The above strain was sent to the China Center for Type Culture Collection for preservation on January 14, 2025. The taxonomic name: Aeromonas caviae Se01, preservation number: CCTCC NO: M2025128, address: Wuhan University, Wuhan, China.

[0012] The above-mentioned Aeromonas caviae, a Gram-positive bacterium, has cells in the shape of short rods, with cell sizes of 0.2-0.4×0.5-0.8 μm. The colonies on the LB solid medium are milky white and opaque; when in the LB medium containing 5 mmol / L sodium selenite, it shows strong reducibility to inorganic selenium, and the colonies are bright red.

[0013] The protection scope of the present invention includes:

[0014] The fermentation broth obtained by fermenting Aeromonas caviae Se01 in an inorganic selenium medium, and the fermentation broth contains viable Aeromonas caviae Se01 and nano selenium.

[0015] A composition, comprising Aeromonas caviae Se01 and / or the above-mentioned fermentation broth.

[0016] The application of Aeromonas caviae Se01, the above-mentioned fermentation broth or the above-mentioned composition in reducing inorganic selenium.

[0017] The application of Aeromonas caviae Se01, the above-mentioned fermentation broth or the above-mentioned composition in preparing biological nano selenium.

[0018] The application of Aeromonas caviae Se01, the above-mentioned fermentation broth or the above-mentioned composition in preparing nano elemental selenium.

[0019] The application of Aeromonas caviae Se01, the above-mentioned fermentation broth or the above-mentioned composition in the cultivation of selenium-rich plants.

[0020] The application of Aeromonas caviae Se01, the above-mentioned fermentation broth or the above-mentioned composition in preparing selenium-rich cultivation preparations.

[0021] For the above-mentioned application, preferably, the plant is Brassica napus.

[0022] For the above-mentioned fermentation broth, preferably, its preparation process is to inoculate Aeromonas caviae Se01 into a medium with an inorganic selenium content of 2-800 mmol / L for fermentation.

[0023] For the above-mentioned fermentation broth, preferably, the inorganic selenium content is 2-200 mmol / L.

[0024] For the above-mentioned fermentation broth, preferably, the inorganic selenium content is 2-11 mmol / L.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) The present invention first provides a strain of Aeromonas caviae with the ability to reduce inorganic selenium.

[0027] (2) The Aeromonas caviae provided by the present invention has both a high selenium tolerance level and a high selenium conversion efficiency, and can be cultured in a culture medium with a high selenium content (800 mmol / L). Usually, the growth of microorganisms in the environment is inhibited or directly killed at this concentration. In addition, during the cultivation of Aeromonas caviae, a high-efficiency inorganic selenium conversion rate can be achieved. Compared with physical and chemical methods for synthesizing nano-selenium, the production cost is lower, which is suitable for popularization.

[0028] (3) The nano-selenium nutrient solution provided by the present invention is obtained by transformation with Aeromonas caviae. Most of it is regular nano-selenium particles, and the organic substances such as carbohydrates, polysaccharides, and proteins attached to its surface effectively improve the adhesion ability. During the process of selenium-rich agricultural production, it can maintain a high biological utilization rate. In addition, compared with nano-selenium synthesized by physical and chemical methods, due to the organic matter coating of biological nano-selenium, its biological stability is higher, it is not easy to accumulate and inactivate, and it can maintain its biological activity for a long time, which can greatly reduce the frequency of repeated application during the agricultural production process and reduce the production cost of selenium-rich agriculture.

[0029] (4) Compared with traditional chemical selenium fertilizers, biological nano-selenium is synthesized through microbial metabolism, which is more environmentally friendly and will not cause environmental pollution of soil and rivers. Its high biological safety has broad development and application prospects in selenium-rich agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Transmission electron microscopy observation and energy spectrum analysis of the bacterial morphology of Aeromonas caviae ( Aeromonas caviae ) Se01;

[0031] Wherein: A and B: are respectively the observation of bacterial morphology and energy spectrum analysis on the surface of bacteria after culturing in normal LB medium for 48 h; C and D: are respectively the observation of bacterial morphology and surface energy spectrum analysis after culturing in LB medium containing 5 mM sodium selenite for 48 h. DETAILED DESCRIPTION OF THE INVENTION

[0032] The technical solutions described in the present invention are all conventional solutions unless otherwise specified; the reagents or materials are all from commercial channels unless otherwise specified. In the examples of the present invention, sodium selenite (i.e., selenite) is taken as an example to illustrate the reduction ability of Aeromonas caviae ( Aeromonas caviae ) Se01 to inorganic selenium; in fact, Aeromonas caviae ( Aeromonas caviae ) Se01 has a high reduction ability to other forms of inorganic selenium, such as selenate. Due to space limitations, the examples of the present invention will not be elaborated further. Example 1:

[0033] Isolation and purification of Aeromonas caviae ( Aeromonas caviae ) Se01:

[0034] At the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, the roots of rapeseed growing in Hoagland nutrient solution containing 40 μmol / L sodium selenite were sampled. After rinsing the surface culture solution with sterile ultrapure water, the surface moisture was blotted dry with sterile filter paper. The roots of rapeseed were placed in a conical flask containing 50 mL of sterile PBS solution and subjected to ultrasonic disruption treatment in an ultrasonic disruptor under ice-water bath conditions. The working power of the above ultrasonic disruption was 40% (about 400 w), with a 30 s interval for every 1 min of work, and the ultrasonic disruption duration was 30 min. Subsequently, the cells in the suspension after disruption were collected by centrifugation and added to LB medium for enrichment culture for 12 h. A certain amount of the culture solution was taken for gradient dilution and spread on a separation plate (LB solid medium containing 5 mmol / L sodium selenite), and incubated at 28 °C for 48 h. The red colonies were picked, which were the strains initially screened with selenium conversion efficiency. After several times of separation and purification, several purified colonies were obtained, and they were respectively subjected to Gram staining and microscopic observation. Amplification was carried out using the universal primers for bacterial identification 16S rDNA (the primer pair sequences were: forward F primer: 5’-AGTTTGATCMTGGCTCAG-3’; reverse R primer: 5’-GGTTACCTTGTTACGACTT-3’), and bacterial identification was carried out after sequencing and comparison. Among them, strain Se01 was identified as Aeromonas caviae ( Aeromonas caviae ).

[0035] The above strain was sent to the China Center for Type Culture Collection on January 14, 2025, and the taxonomic name was: Aeromonas caviae Se01, with the preservation number: CCTCC NO: M2025128, address: Wuhan University.

[0036] The above Aeromonas caviae, a Gram-positive bacterium, has cells in the shape of short rods, with cell size of 0.2 - 0.4 × 0.5 - 0.8 μm. The colonies on the LB solid medium are milky white and opaque; when in the LB medium containing 5 mmol / L sodium selenite, it shows strong reducibility to inorganic selenium, and the colonies are bright red. Example 2:

[0037] Application of Aeromonas caviae ( Aeromonas caviae ) Se01 in the reduction of inorganic selenium:

[0038] (1) Strain activation: Aeromonas caviae ( Aeromonas caviae ) was inoculated on LB solid medium and incubated in an incubator at 28 °C for 24 h to obtain the activated strain;

[0039] (2) Preparation of active bacterial liquid: The activated strain was inoculated into LB liquid medium and cultured in a constant temperature shaker at 28 °C and 200 rpm for 16 h until OD600 With a value of 1.2, an active bacterial solution is obtained;

[0040] (3) Inoculation: The active bacterial solution is respectively inoculated into a normal LB liquid medium and an LB liquid medium containing inorganic selenium. The volume ratio of the active bacterial solution to the culture solution is 1:1000, and it is cultured in a constant temperature shaker at 28 °C with 200 rpm for 48 h; the LB liquid medium containing inorganic selenium includes: 10 g / L of tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride; sodium selenite is added as inorganic selenium, with a pH value of 7.0, and the rest is water. The concentration of sodium selenite is 5 mmol / L;

[0041] (4) Electron microscopy observation: After the inoculated and cultured bacterial solution is centrifuged to collect the supernatant, it is washed with sterile ultrapure water and then fixed with a fixative and observed under a scanning electron microscope. The results are as Figure 1 shown. Through scanning electron microscopy observation and energy spectrum analysis, Aeromonas caviae ( Aeromonas caviae ) Se01 can reduce sodium selenite and synthesize uniformly sized circular nano-selenium particles when cultured in an LB liquid medium containing inorganic selenium. Example 3:

[0042] Application of Aeromonas caviae ( Aeromonas caviae ) Se01 in the biotransformation of inorganic selenium:

[0043] (1) Strain activation: Aeromonas caviae ( Aeromonas caviae ) is inoculated on an LB solid medium and cultured in an incubator at 28 °C for 24 h to obtain an activated strain;

[0044] (2) Fermentation broth preparation: The activated strain is inoculated into an LB liquid medium and cultured in a constant temperature shaker at 28 °C with 200 rpm for 16 h until the OD 600 value reaches 1.2 to obtain an active fermentation broth;

[0045] (3) Inoculation and culture: The fermentation broth is added to an LB liquid medium containing different concentrations of inorganic selenium. The volume ratio of the fermentation broth to the inorganic selenium-containing medium is 1:500, and it is fermented and cultured in a constant temperature shaker at 28 °C with 200 rpm for 48 h to obtain the product; the inorganic selenium content in the medium before and after fermentation and the nano-selenium content in the fermentation broth after fermentation are detected, and the results are shown in Table 1;

[0046] The inorganic selenium-containing medium includes: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, sodium selenite is added as inorganic selenium, the pH value is 7.0, and the rest is water. The concentrations of the above sodium selenite are 2.02 mmol / L, 5.06 mmol / L, 10.43 mmol / L, 20.66 mmol / L, 39.21 mmol / L, 93.41 mmol / L, 206.16 mmol / L, 416.22 mmol / L, 513.63 mmol / L, 592.95 mmol / L, 802.38 mmol / L respectively.

[0047] Table 1 Identification of nano-selenium content after 48 hours of fermentation at different inorganic selenium concentrations

[0048] 。

[0049] The measurement of nano-selenium refers to the reference source: Biswas, K. C., L. L. Barton, et al. (2011). "A novel method for the measurement of elemental selenium produced by bacterial reduction of selenite." Journal of Microbiological Methods 86(2):140-144.

[0050] The conversion rate formula of the consumed inorganic selenium after 48 hours of fermentation is as follows:

[0051] 。

[0052] From the above results, it can be seen that ( Aeromonas caviae Se01) can tolerate 800 mmol / L of inorganic selenium in the medium and continuously maintain the reduction activity; at the same time, in the fermentation product, nano-selenium is the main component. When the content of inorganic selenium in the medium is 2-200 mmol / L, more than 70% of the fermentation broth is nano-selenium. When the content of inorganic selenium in the medium is 2-11 mmol / L, more than 96% of the fermentation broth is nano-selenium.

[0053] Example 4:

[0054] Aeromonas caviae ( Aeromonas caviae ) Se01 is used to prepare bio-nano-selenium:

[0055] The fermentation broth at 48 h in the fermentation group with a selenium concentration of 5.04 mmol / L in Table 1 of Example 3 above was centrifuged to remove the supernatant, and the precipitate was washed 3 times with ultrapure water. The cells were frozen and dehydrated using a freeze dryer (Scientz-30ND) to convert the strain from a liquid state to a dry powder form, obtaining nano-selenium dry powder, which was stored in a -80 °C refrigerator. Particle size and Zeta potential analysis of the nano-selenium dry powder showed that the bio-nano-selenium particles prepared by Aeromonas caviae ( Aeromonas caviae ) Se01 were of uniform size, with an average diameter of 176 nm and a Zeta potential of -18.9 ± 0.5 mV, having good biological activity and showing good development and application prospects as a new biological material.

[0056] Example 5:

[0057] Application of bio-nano-selenium prepared by Aeromonas caviae ( Aeromonas caviae ) Se01 in rapeseed cultivation:

[0058] The nano-selenium dry powder in Example 4 above was diluted with water to a nano-selenium nutrient solution mother liquor with a concentration of 1 mmol / L, which was used for hydroponic cultivation of Brassica napus. When the rapeseed grew to the five-leaf stage, the nano-selenium nutrient solution mother liquor and sodium selenite mother liquor were added respectively to keep the final selenium concentration in the nutrient solution at 40 μmol / L. After culturing for one week, the rapeseed seedlings were collected, and the selenium forms in the above-ground part and roots were determined. Table 2 shows the selenium forms and contents in the rapeseed seedlings after culturing for one week.

[0059] Table 2 Selenium-enriched rapeseed cultivation

[0060] .

[0061] It can be found that compared with adding sodium selenite at the same concentration, by adding the bio-nano-selenium prepared in the present invention, the proportion of methylselenocysteine in rapeseed seedlings can be effectively increased, the organic selenium components retained in the roots are less, and the total selenium content, organic content and methylselenocysteine content in the edible above-ground part are all significantly increased, showing broad application prospects in selenium-enriched agricultural production.

Claims

1. An isolated strain of Aeromonas caviae ( Aeromonas caviae )Se01, the preservation number of the Aeromonas caviae is CCTCC NO: M2025128.

2. The fermentation broth obtained by fermenting the Aeromonas caviae Se01 according to claim 1 in an inorganic selenium culture medium, wherein the fermentation broth contains live Aeromonas caviae Se01 bacteria and nano-selenium.

3. The fermentation broth according to claim 2, characterized in that: The preparation method is to inoculate the Aeromonas caviae Se01 described in claim 1 into a culture medium with an inorganic selenium content of 2-800 mmol / L and ferment it.

4. A composition comprising the Aeromonas caviae Se01 according to claim 1 and / or the fermentation broth according to claim 2.

5. Use of Aeromonas caviae Se01 according to claim 1, the fermentation broth according to claim 2 or the composition according to claim 3 in reducing inorganic selenium.

6. Use of Aeromonas caviae Se01 according to claim 1, the fermentation broth according to claim 2 or the composition according to claim 3 in the preparation of bio-nanoselenium.

7. Use of Aeromonas caviae Se01 according to claim 1, the fermentation broth according to claim 2 or the composition according to claim 3 in the preparation of nano-elemental selenium.

8. Use of Aeromonas caviae Se01 according to claim 1, the fermentation liquid according to claim 2 or the composition according to claim 3 in the cultivation of selenium-rich plants, wherein the plant is Brassica napus.

9. Use of Aeromonas caviae Se01 according to claim 1, the fermentation liquid according to claim 2 or the composition according to claim 3 in preparing a selenium-enriched planting preparation, wherein the plant is Brassica napus.

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