Separated guinea pig aeromonas and application of separated guinea pig aeromonas in nano-selenium preparation

Abstract

The invention relates to the technical field of biology, and discloses separated Aeromonas caviridis and application of the separated Aeromonas caviridis in preparation of nano-selenium, and the preservation number of the Aeromonas caviridis is CCTCC (China Center For Type Culture Collection) NO: M2025128. The invention provides the Aeromonas cainea with both selenium tolerance and selenium reducing capacity for the first time, and the Aeromonas cainea can tolerate a high-concentration selenium salt culture solution and synthesize biological nano-selenium. A nano-selenium nutrient solution can be prepared from the Aeromonas caviridis Se01 provided by the invention, is applied to selenium-enriched agricultural planting, has a remarkable selenium biological enhancement effect on oilseed rape, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial application and biosynthesis of nano-selenium, and specifically to an isolated Aeromonas caviae and its application in the preparation of nano-selenium. The applicant has isolated, purified and identified the strain adsorbed on the root system of rapeseed, and obtained a strain that can efficiently reduce selenite and synthesize biological nano-selenium. Background Art

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

[0003] At present, the selenium-rich level of crops is mainly improved by foliar spraying or soil application of selenium fertilizer. Sodium selenite is the main selenium fertilizer, but different crops have great differences in their absorption and conversion capabilities, and often need to be applied frequently to achieve an increase in selenium nutrition levels. The application of inorganic selenium fertilizers in agricultural practice will lead to high production costs, and will also bring varying degrees of soil and river selenium pollution. With the development of science and technology, biological nano-selenium has attracted much attention. The main reason is that nano-selenium has a higher surface area-to-volume ratio and is easier for plants to absorb; compared with nano-selenium synthesized by traditional physical or chemical means, biological nano-selenium has a more regular shape, and its surface amino acid modification gives it higher biological activity and stability; in addition, biological nano-selenium has a higher adhesion ability and does not require multiple applications. It 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-enriched agriculture and environmental governance.

[0004] As a key raw material for the synthesis of biological nano-selenium, inorganic selenium usually has high cytotoxicity, inhibiting the growth of many microorganisms and even causing 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 that have both selenium tolerance and selenium reduction.

[0005] Rapeseed is a Brassica crop that has the ability to efficiently absorb, transform and enrich selenium. It is regarded as an excellent bioreactor for selenium and has attracted 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 40μM sodium selenite. Inorganic selenium can be converted into biological nano-selenium through liquid culture. The prepared biological nano-selenium can be applied to the selenium-enriched cultivation process of rapeseed, and has achieved good results, with 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 ), can efficiently synthesize biological nano-selenium and be applied in 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 bio-nanoselenium, which is simple, efficient, convenient and suitable for large-scale promotion.

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

[0009] In order to achieve the above object, the present invention adopts the following technical measures: The applicant collected rapeseed roots grown in a hydroponic nutrient solution containing 40 μM sodium selenite at the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, treated them with an ultrasonic disruptor, and added them to LB culture medium for enrichment culture. A certain amount of culture solution was spread on a separation plate (LB solid culture medium containing 2 mmol / L sodium selenite), and after 48 hours of culture, red colonies were picked. After several isolation and purifications, several clones were obtained, and the isolated strains were subjected to Gram staining and microscopic examination to observe their bacterial morphological characteristics. The 16S rDNA universal primers for bacterial species identification (the primer pair sequences are: forward F primer: 5'-AGTTTGATCMTGGCTCAG-3'; reverse R primer: 5'-GGTTACCTTGTTACGACTT-3') were used for amplification, and the bacterial species were identified after sequencing comparison. Among them, the Se01 strain was identified as Aeromonas caviae ( Aeromonas caviae ).

[0010] The above strains were sent to China Center for Type Culture Collection for preservation on January 14, 2025, and the classification and naming are as follows: Aeromonas caviae Se01, deposit number: CCTCC NO: M2025128, address: Wuhan University, Wuhan, China.

[0011] The above-mentioned Aeromonas caviae is a Gram-positive bacterium with short rod-shaped cells and a 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 exhibits strong reducing properties to inorganic selenium and the colonies are bright red.

[0012] The protection scope of the present invention includes: The fermentation liquid is obtained by fermenting Aeromonas caviae Se01 in an inorganic selenium culture medium, and the fermentation liquid contains live bacteria of Aeromonas caviae Se01 and nano-selenium.

[0013] A composition comprises Aeromonas caviae Se01 and / or the fermentation broth.

[0014] Application of Aeromonas caviae Se01, the fermentation broth or the composition in reducing inorganic selenium.

[0015] Application of Aeromonas caviae Se01, the fermentation broth or the composition in the preparation of biological nano-selenium.

[0016] Application of Aeromonas caviae Se01, the fermentation broth or the composition in the preparation of nano-elemental selenium.

[0017] Application of Aeromonas caviae Se01, the fermentation liquid or the composition in the cultivation of selenium-rich plants.

[0018] Application of Aeromonas caviae Se01, the fermentation liquid or the composition in the preparation of selenium-enriched planting preparations.

[0019] In the above application, preferably, the plant is Brassica napus.

[0020] The fermentation broth described above is preferably prepared by inoculating Aeromonas caviae Se01 into a culture medium having an inorganic selenium content of 2-800 mmol / L and fermenting the culture medium.

[0021] The fermentation broth described above preferably has an inorganic selenium content of 2-200 mmol / L.

[0022] The fermentation broth described above preferably has an inorganic selenium content of 2-11 mmol / L.

[0023] Compared with the prior art, the present invention has the following advantages: (1) The present invention provides for the first time a strain of Aeromonas caviae that has the ability to reduce inorganic selenium.

[0024] (2) The Aeromonas caviae provided by the present invention has both high selenium tolerance and selenium conversion efficiency, and can be cultured in a culture medium with a high selenium concentration (800 mmol / L), while microorganisms in the environment are usually inhibited from growing or directly killed at this concentration. In addition, during the culture of Aeromonas caviae, a high-efficiency inorganic selenium conversion rate can be achieved. Compared with the synthesis of nano-selenium by physical and chemical means, the production input cost is lower and suitable for promotion.

[0025] (3) The nano-selenium nutrient solution provided by the present invention is obtained by transformation with the help of Aeromonas caviae, and most of it is regular nano-selenium particles. The organic matter such as carbohydrates, polysaccharides, and proteins attached to its surface effectively improves its adhesion ability, and can maintain a high bioavailability during the selenium-rich agricultural production process. In addition, compared with nano-selenium synthesized by physical and chemical means, since biological nano-selenium is coated with organic matter, it has higher biological stability, is not easy to accumulate and inactivate, and can maintain its biological activity for a long time, which can greatly reduce the frequency of repeated application in the agricultural production process and reduce the production cost of selenium-rich agriculture.

[0026] (4) Compared with traditional chemical selenium fertilizers, bio-nanoselenium is synthesized through microbial metabolism and is more environmentally friendly. It will not cause environmental pollution to the soil and rivers. Its high biosafety has broad development and application prospects in selenium-rich agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Aeromonas caviae ( Aeromonas caviae )Transmission electron microscopy observation and energy spectrum analysis of Se01 bacterial morphology; Among them: A and B: the bacterial morphology observation and the energy spectrum analysis of the bacterial surface after 48 hours of culture in normal LB medium; C and D: the bacterial morphology observation and the energy spectrum analysis of the surface after 48 hours of culture in LB medium containing 5mM sodium selenite. DETAILED DESCRIPTION

[0028] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions; the reagents or materials described, unless otherwise specified, are all from commercial channels. The present embodiment takes sodium selenite (i.e., selenite) as an example to illustrate the Aeromonas caviae ) Se01's ability to reduce inorganic selenium; in fact, Aeromonas caviae ( Aeromonas caviae ) SeO1 has a highly efficient reducing ability to other forms of inorganic selenium, such as selenate, which will not be described in detail in the embodiments of the present invention due to space limitations. Embodiment 1:

[0029] Aeromonas caviae ( Aeromonas caviae ) Separation and purification of Se01: At the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, samples were collected from the roots of rapeseed grown in Hoagland nutrient solution containing 40 μmol / L sodium selenite. After the surface culture solution was rinsed with sterile ultrapure water, the surface water was dried with sterile filter paper. The roots of rapeseed were placed in a conical flask with 50 mL of sterile PBS solution and ultrasonically disrupted in an ultrasonic disruptor under ice-water bath conditions. The working power of the ultrasonic disruption was 40% (about 400w), with an interval of 30s for every 1min of work, and the ultrasonic disruption time was 30min. Subsequently, the suspended bacterial cells after disruption were collected by centrifugation and added to LB medium for enrichment culture for 12h. A certain amount of culture solution was taken for gradient dilution and coated on a separation plate (LB solid medium containing 5 mmol / L sodium selenite), and cultured at 28℃ for 48h. The red colonies were picked, which were the strains with selenium conversion efficiency in the initial screening. After several separation and purifications, several purified colonies were obtained, which were subjected to Gram staining and microscopic observation. The strain was amplified using the universal 16S rDNA primers for bacterial species identification (primer pair sequences were: forward F primer: 5'-AGTTTGATCMTGGCTCAG-3'; reverse R primer: 5'-GGTTACCTTGTTACGACTT-3'), and the strain was identified after sequencing. The Se01 strain was identified as Aeromonas caviae ( Aeromonas caviae ).

[0030] The above strains were sent to China Center for Type Culture Collection on January 14, 2025 and were classified and named as: Aeromonas caviae Se01, deposit number: CCTCC NO: M2025128, address: Wuhan University.

[0031] The above-mentioned Aeromonas caviae is a Gram-positive bacterium with short rod-shaped cells and a 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 exhibits strong reducing properties to inorganic selenium and the colonies are bright red. Embodiment 2:

[0032] Aeromonas caviae ( Aeromonas caviae )Application of Se01 in inorganic selenium reduction: (1) Strain activation: Aeromonas caviae ( Aeromonas caviae ) was inoculated on LB solid medium and placed in a 28°C incubator for 24 hours to obtain activated strains; (2) Preparation of active bacterial liquid: The activated bacteria were inoculated into LB liquid medium and cultured in a constant temperature shaker at 28°C and 200 rpm for 16 h until the OD 600 The value is 1.2, and active bacterial solution is obtained; (3) Inoculation: The active bacterial liquid was inoculated into a normal LB liquid culture medium and an LB liquid culture medium containing inorganic selenium, respectively, with the volume ratio of the active bacterial liquid to the culture medium being 1:1000, and the culture was cultured at 28°C constant temperature shaker at 200 rpm for 48 h; the LB liquid culture medium containing inorganic selenium included: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride; sodium selenite was added as inorganic selenium, the pH value was 7.0, and the rest was water, and the concentration of sodium selenite was 5 mmol / L; (4) Electron microscopic observation: The bacterial culture was centrifuged and the supernatant was collected. After washing with sterile ultrapure water, the culture was fixed with a fixative and observed under a scanning electron microscope. The results are as follows: Figure 1 As shown, by scanning electron microscopy and energy spectrum analysis, Aeromonas caviae ( Aeromonas caviae ) Se01 was cultured in LB liquid medium containing inorganic selenium and was able to reduce sodium selenite to synthesize round nano-selenium particles of uniform size. Embodiment 3:

[0033] Aeromonas caviae ( Aeromonas caviae )Application of Se01 in the biotransformation of inorganic selenium: (1) Strain activation: Aeromonas caviae ( Aeromonas caviae ) was inoculated on LB solid medium and placed in a 28°C incubator for 24 hours to obtain activated strains; (2) Preparation of fermentation broth: The activated strain was inoculated into LB liquid medium and cultured at 28°C constant temperature shaker, 200 rpm for 16 h until OD 600 value 1.2, and active fermentation broth was obtained; (3) Inoculation culture: the fermentation broth was added to LB liquid culture medium containing different concentrations of inorganic selenium, the volume ratio of the fermentation broth to the culture medium containing inorganic selenium was 1:500, and the culture was carried out in a constant temperature shaker at 28°C and 200 rpm for 48 h. The inorganic selenium content in the culture medium before and after fermentation, and the nano-selenium content in the fermentation broth after fermentation were detected. The results are shown in Table 1. The inorganic selenium-containing culture medium comprises: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, sodium selenite added as inorganic selenium, a pH value of 7.0, and the rest is water, and the concentrations of the 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, and 802.38 mmol / L, respectively.

[0034] Table 1 Identification of nano-selenium content after fermentation for 48 hours at different inorganic selenium concentrations .

[0035] Reference for determination of nano-selenium: Biswas, KC, LL 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. The conversion rate of consumed inorganic selenium after 48 hours of fermentation is calculated using the following formula: .

[0036] From the above results, we can see that ( Aeromonas caviae ) Se01, in the culture medium, can tolerate 800mmol / L of inorganic selenium and continue to maintain reducing activity; at the same time, in the fermentation product, nano-selenium is the main component. When the content of inorganic selenium in the culture medium is 2-200mmol / L, more than 70% of the fermentation liquid is nano-selenium. When the content of inorganic selenium in the culture medium is 2-11mmol / L, more than 96% of the fermentation liquid is nano-selenium.

[0037] Embodiment 4: Aeromonas caviae ( Aeromonas caviae )Se01 preparation of biological nano-selenium: The fermentation broth of 48h in the fermentation group with a selenium concentration of 5.04 mmol / L in Table 1 of Example 3 was centrifuged to remove the supernatant, and the precipitate was washed with ultrapure water three times. The bacteria were frozen and dehydrated using a freeze dryer (Scientz-30ND) to transform the strain from liquid to dry powder to obtain nano-selenium dry powder, which was stored in a -80°C refrigerator. The particle size and Zeta potential analysis of the nano-selenium dry powder showed that Aeromonas caviae ( Aeromonas caviae ) The size of biological nano-selenium particles prepared by Se01 is consistent, with an average diameter of 176nm and a Zeta potential of -18.9±0.5mV. It has good biological activity and has good development and application prospects as a new type of biomaterial.

[0038] Embodiment 5: Aeromonas caviae ( Aeromonas caviae )Application of Se01 in preparing bio-nanoselenium in rapeseed cultivation: The nano-selenium dry powder in Example 4 was diluted with water to a nano-selenium nutrient solution with a concentration of 1 mmol / L, and used for hydroponic cultivation of Brassica napus. When the rapeseed grew to the five-leaf stage, the nano-selenium nutrient solution and the sodium selenite solution were added respectively to maintain the final concentration of selenium in the nutrient solution at 40 μmol / L. After one week of cultivation, the rapeseed seedlings were collected and the selenium forms in the aboveground part and the root were determined. Table 2 shows the selenium forms and contents in the seedlings after one week of cultivation.

[0039] Table 2 Selenium-enriched rapeseed planting .

[0040] It can be found that compared with adding sodium selenite at the same concentration, adding the biological nano-selenium prepared by the present invention can effectively increase the proportion of methylselenocysteine ​​in rapeseed seedlings, less organic selenium components are accumulated in the root system, and the total selenium content, organic content and methylselenocysteine ​​content of the edible aboveground parts are significantly improved, and it has broad application prospects in selenium-rich 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.

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 plant planting preparation.

10. The use according to claim 8 or 9, characterized in that: The plant is Brassica napus.

Images