Accelerator for microalgae synthesis of nano-selenium, and method for microalgae synthesis of nano-selenium

Abstract

The present application belongs to the field of biomass resource utilization and bioenergy, and in particular relates to an accelerator for microalgae to synthesize nano-selenium and a method for microalgae to synthesize nano-selenium. The present application provides an accelerator for microalgae to synthesize nano-selenium, a flavonoid compound, sodium selenite and a solvent. The flavonoid compound is selected from one or more of quercetin, rutin, Rhus chinensis leaf extract and lotus leaf extract. The present application provides an accelerator for microalgae to synthesize nano-selenium and a method for microalgae to synthesize nano-selenium, the accelerator can significantly promote the conversion efficiency of microalgae to inorganic selenium and accumulate nano-selenium; and the method for synthesizing nano-selenium is highly efficient and low-cost, and the process for producing nano-selenium is green and environmentally friendly, and has broad industrial application prospects.

Description

Technical Field

[0001] The present application belongs to the field of biomass resource utilization and bioenergy, and in particular relates to a promoter for synthesizing nano-selenium by microalgae and a method for synthesizing nano-selenium by microalgae. Background Art

[0002] Selenium is an essential trace element for the human body, playing a crucial role in antioxidant defense and cancer prevention. According to WHO data, selenium deficiency or deficiency is prevalent in over 40 countries, making it a widespread problem worldwide. The "Atlas of Local Diseases and Environmental Factors of the People's Republic of China" indicates that a low-selenium belt stretching from the three northeastern provinces to the Yunnan-Guizhou Plateau, covering over 72% of the country's total area, is inhabited by a region with a low selenium content, with 30% of this area experiencing severe selenium deficiency. Inadequate dietary selenium intake leads to a variety of diseases, such as Keshan disease. Therefore, selenium supplementation is crucial. As mentioned above, selenium deficiency can lead to a range of illnesses, but excessive selenium intake can also cause neurological disorders, nail and hair loss, and other abnormalities. Therefore, while selenium supplementation is crucial, it's also crucial to ensure that it's done in a healthy, safe, and scientifically sound manner.

[0003] Dietary selenium supplementation is the most economical, safe, and effective approach. Selenium primarily exists in inorganic and organic forms in various selenium supplement products, such as sodium selenite. However, these products have low bioavailability and are prone to selenium poisoning, peroxidation, and potential pollution. Organic selenium, such as selenomethionine, can also be used as a dietary supplement. However, selenomethionine can replace normal methionine for protein synthesis, posing a risk of toxicity if consumed and accumulated over a long period of time.

[0004] Studies have shown that nanoselenium has high bioabsorption, high safety, and strong antioxidant capacity. Therefore, nanoselenium has attracted great interest as a dietary supplement. Nanoselenium can also act as an immunomodulator, stimulating the body's immune system, and is therefore considered a product with great biological and pharmaceutical benefits. Furthermore, nanoselenium also possesses the novel physical and chemical properties of nanomaterials, and therefore has industrial applications in many areas, such as optoelectronic materials and semiconductors. Currently, the production of nanoselenium is mainly achieved through chemical synthesis. The chemical synthesis process of nanoselenium is often accompanied by chemical contamination of the product. In order to produce selenium products in a more environmentally friendly way, the use of organisms to convert inorganic selenium into organic selenium and nanoselenium with low toxicity and safe for consumption has become a hot topic.

[0005] Many plants and microorganisms accumulate selenium, and current selenium supplements primarily rely on selenium-rich plants and yeast. Different yeast strains vary significantly in their resistance to inorganic selenium, and thus their ability to absorb and convert it. This results in a low conversion rate of organic selenium, and consequently, poor absorption and utilization by the human body. Furthermore, a certain amount of unconverted inorganic selenium can easily lead to selenium poisoning. Summary of the Invention

[0006] In view of this, the present application provides an accelerator for the synthesis of nano-selenium by microalgae and a method for the synthesis of nano-selenium by microalgae. The accelerator can significantly promote the conversion efficiency of inorganic selenium by microalgae and accumulate nano-selenium. Moreover, the method for synthesizing nano-selenium is highly efficient and low-cost, and the production process of nano-selenium is green and environmentally friendly, and has broad industrial application prospects.

[0007] In a first aspect, the present application provides an accelerator for synthesizing nano-selenium by microalgae, comprising flavonoid compounds, sodium selenite and a solvent.

[0008] Preferably, the mass ratio of the flavonoid compound to the sodium selenite is in the range of 1:40 to 1:1000.

[0009] More preferably, the mass ratio of the flavonoid compound to the sodium selenite is 1:1000.

[0010] Preferably, the flavonoids are selected from one or more of quercetin, rutin, Rhus chinensis leaf extract and lotus leaf extract.

[0011] More preferably, the flavonoid is quercetin.

[0012] Specifically, the solvent used in the promoter of the present application is an organic solvent that has little or no effect on the growth of microalgae.

[0013] Preferably, the solvent is selected from one or more of polyethylene glycol, ethanol, acetic acid and ethanol.

[0014] More preferably, the solvent is polyethylene glycol.

[0015] The second aspect of the present application provides a method for synthesizing nano-selenium by microalgae, comprising the following steps:

[0016] Step 1: obtaining microalgae in the stationary phase, mixing a promoter with a culture medium containing the microalgae to obtain treated microalgae; wherein the promoter includes the promoter described above;

[0017] Step 2: extracting nano-selenium from the treated microalgae.

[0018] Specifically, in addition to extracting nano-selenium from the treated microalgae, active substances such as astaxanthin can also be extracted from the treated microalgae.

[0019] Preferably, in step 1, the volume percentage of the flavonoid compound in the culture medium is Specifically, the flavonoid compound is added to the culture medium in step 1) in an amount of 12.5-50 μg / L.

[0020] More preferably, the amount of the flavonoid compound added to the culture medium in step 1) is 25 μg / L.

[0021] Preferably, in step 1, the mixed culture time is 1 to 7 days.

[0022] More preferably, the mixed culture time is 5 days.

[0023] Preferably, in step 1, the microalgae are selected from one or more of Haematococcus pluvialis, Dunaliella salina or Chlorella vulgaris.

[0024] More preferably, the microalgae is Haematococcus pluvialis.

[0025] Preferably, in step 1, the density of the microalgae in the plateau phase is 1×10 6 -2×10 6 pieces / mL.

[0026] More preferably, the density of the microalgae in the plateau phase is 1.5×10 6 pieces / mL.

[0027] Preferably, in step 2, the extraction specifically comprises: separating and collecting microalgae from the culture medium of step 1, and then extracting nano-selenium from the microalgae.

[0028] Specifically, the method for extracting nano-selenium is an existing conventional method for extracting nano-selenium from plant cells.

[0029] The present application unexpectedly discovered that flavonoids have the effect of promoting the synthesis of nano-selenium in microalgae, opening up a promoter for promoting the accumulation of nano-selenium in microalgae and a method for green production of nano-selenium. The present application provides a method for producing nano-selenium with low cost and convenient operation. Plant-derived flavonoids can promote microalgae cells to convert inorganic selenium into nano-selenium, increasing the potential application value of microalgae cells and creating a new model for safe and efficient production of nano-selenium. Experimental data show that flavonoids can significantly promote the production of nano-selenium in microalgae. Compared with the nano-selenium content in normally cultured microalgae, the nano-selenium content of microalgae in the flavonoid-treated group was significantly increased, and the total nano-selenium content in the flavonoid-treated group increased from 0.7% to 3.85% of the dry weight. The results show that flavonoids put the microalgae in a state of stress, filling the cells with a large number of lipid droplets, and significantly increasing the content of nano-selenium and astaxanthin. This shows that flavonoids can not only promote the production of nano-selenium in microalgae, but also increase the content of high-value-added products (such as astaxanthin) in microalgae. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art.

[0031] Figure 1 The figure shows the conversion efficiency of sodium selenite into nano-selenium by Haematococcus pluvialis provided in Example 1 of the present application;

[0032] Figure 2 1. The morphological structure of nano-selenium synthesized by Haematococcus pluvialis is observed by electron microscopy according to Example 1 of the present application;

[0033] Figure 3 The changes in carbohydrate content in Haematococcus pluvialis cells provided in Example 1 of the present application are shown;

[0034] Figure 4 1 and 2 show the changes in astaxanthin content in Haematococcus pluvialis cells provided in Example 1 of the present application. DETAILED DESCRIPTION

[0035] The present application provides an accelerator for synthesizing nano-selenium by microalgae and a method for synthesizing nano-selenium by microalgae, which are used to solve the technical defect of low conversion rate of nano-selenium synthesized by plants and microorganisms in the prior art.

[0036] The following is a clear and complete description of the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0037] The reagents used in the following examples are all commercially available.

[0038] Example 1

[0039] The present application provides an experiment of mixing a promoter with a culture medium containing stationary-stage Haematococcus pluvialis, specifically comprising:

[0040] 1. Prepare an accelerator for the synthesis of nano-selenium by microalgae by mixing quercetin, sodium selenite and polyethylene glycol to obtain the accelerator, wherein the mass ratio of quercetin to sodium selenite is 1:1000.

[0041] 2. Cultivate to the plateau stage (inoculate Haematococcus pluvialis into fresh culture medium, the initial concentration of Haematococcus pluvialis is 10 4cells / mL, and entered the plateau phase on the 16th day of culture) were divided into two groups, one group was the control group (only sodium selenite was added to the culture medium without quercetin, the concentration of sodium selenite in the culture medium was 25 mg / L, and this group was marked as Ctr(Se)), and the other group was the promoter group (quercetin was added to the culture medium with the same concentration of sodium selenite, and the concentration of quercetin in the culture medium was 25 μg / L, and this group was marked as Se+FA), and then cultured for another 5 days. The Haematococcus pluvialis of the control group and the promoter group with different culture times were taken, separated by centrifugation, and the supernatant culture fluid was taken for quantitative analysis using ICP-MS (inductively coupled plasma mass spectrometry) to determine the content of sodium selenite in the culture medium, thereby determining the conversion efficiency of Haematococcus pluvialis to inorganic selenium. The results are as follows Figure 1 shown.

[0042] from Figure 1 It can be seen that after adding the accelerator, only a very low selenium content (less than 5%) was detected in the supernatant of the Haematococcus pluvialis culture medium. Therefore, it can be seen that after the Haematococcus pluvialis culture medium was stimulated by the flavonoid quercetin, the sodium selenite added to the Haematococcus pluvialis cells was quickly converted into nano-selenium. The conversion rate reached 94% on the second day after addition, and remained stable for several days thereafter, reaching an overall conversion rate of 95%. The addition of the accelerator significantly increased the efficiency of the Haematococcus pluvialis in converting sodium selenite to nano-selenium. The total nano-selenium content of the Haematococcus pluvialis in the control group and the accelerator group was tested. The results showed that the control group did not detect a significant increase in nano-selenium, remaining at 0.6, while the total nano-selenium content in the accelerator group increased from 0.7% of dry weight to 3.85%.

[0043] 3. Scanning electron microscopy analysis of the Haematococcus pluvialis algae in the above promoter group was performed. The results are as follows: Figure 2 As shown, Figure 2 The results showed that the accelerator-stimulated Haematococcus pluvialis not only increased the absorption and conversion rate of inorganic selenium, but also clearly revealed the morphology and size of the converted selenium particles within the algal cells through scanning electron microscopy. Analysis showed that the diameters of these selenium particles ranged from 50 to 300 nm, indicating that nano-selenium had been successfully produced within the algal cells.

[0044] 4. Carbohydrate content of Haematococcus pluvialis in the above-mentioned promoter group was tested. The specific steps are as follows: Use a 50mL centrifuge tube to collect the algae solution that has been cultured to the plateau stage after inoculation, and centrifuge at 4000rpm for 3 minutes. Resuspend the collected algae mud in 1mL of sterilized UP water; add 1mL of 5% phenol solution and 5mL of concentrated sulfuric acid to the above-mentioned resuspended algae solution; water bath at 25℃ for 10min, then transfer to a 30℃ water bath and incubate for 20min; take about 150mg of excess glucose and put it in a 60℃ oven to dry for 24 hours, and weigh 100mg. Accurately determine the volume in a 100mL brown volumetric flask to obtain a reference solution; arrange the standard solution diluted in a gradient and the sample to be tested obtained above in a 96-well plate, and measure it with an enzyme reader at a wavelength of 483nm; make a standard curve and use the regression equation to calculate the polysaccharide content in the algae solution. The results are as follows Figure 3 shown. Figure 3 It can be seen that when the promoter stimulated Haematococcus pluvialis to convert selenium, the carbohydrate content in the algae cells decreased significantly. This is because the carbon flow in the algae cells was more used to synthesize lipids and proteins, including selenoproteins.

[0045] 5. The astaxanthin content in the algae cells of the Haematococcus pluvialis of the above-mentioned promoter group was detected. The specific steps are as follows: 100 mL of algae cells at different treatment periods after the platform period were collected in a sterilized 50 mL centrifuge tube in a clean bench, centrifuged at 4000×g for 5 minutes at 4°C, and the supernatant was discarded. The remaining algae mud was collected in a sterilized 1.5 mL EP, centrifuged at 8000 rpm for 1 minute, and the supernatant was discarded; 1 mL of mixed solution (30% methanol and 5% sodium hydroxide) was added to the sample and transferred to a 5 mL EP tube, and ultrasonically crushed in a beaker fixed with ice. The algae were crushed at 90HZ for 15 minutes; 1 mL of mixed solution (30% methanol and 5% sodium hydroxide, treated at 65°C for 15 minutes to remove chlorophyll) was added to the crushed algae, and then washed with sterilized UP water, and the OD was measured. 480 The relative content of astaxanthin was detected by measuring the absorbance value at nm. Figure 4 shown. Figure 4 It can be seen that the detection and analysis of astaxanthin in algal cells after stimulation with quercetin for different days found that compared with the untreated control group, the astaxanthin content in the algal cells treated with quercetin in the above-mentioned promoter group was increased to a certain extent.

[0046] Example 2

[0047] The present application provides an experiment of mixing a promoter with a culture medium containing stationary-stage Haematococcus pluvialis, specifically comprising:

[0048] The method of this embodiment is similar to that of Example 1, except that the quercetin in Example 1 is replaced with lotus leaf extract. The other parameters are consistent with those of Example 1. The results show that the inorganic selenium conversion rate of this embodiment is 90%, and the nano-selenium content is increased by 4 times.

[0049] Example 3

[0050] The present application provides a comparative test of different ratios of promoters on the synthesis of nano-selenium by Haematococcus pluvialis, specifically including:

[0051] 1. Prepare accelerators in different proportions: mix Rhus chinensis leaf extract, sodium selenite and polyethylene glycol to obtain accelerators in different proportions.

[0052] 2. Cultivate to the plateau stage (inoculate Haematococcus pluvialis into fresh culture medium, the initial concentration of Haematococcus pluvialis is 10 4 cells / mL, and entered the plateau phase on the 16th day of culture, were divided into four groups, the first group being a control group (only sodium selenite was added to the culture medium without adding Rhus chinensis leaf extract, and the concentration of sodium selenite in the culture medium was 0.5 mg / L), and the other three groups being promoter groups (divided into promoter groups 1 to 3, i.e., promoters with different ratios were added to the culture medium, the concentration of Rhus chinensis leaf extract in promoter group 1 was 12.5 μg / L, and the concentration of sodium selenite was 0.5 mg / L; the concentration of Rhus chinensis leaf extract in promoter group 2 was 25 μg / L, and the concentration of sodium selenite was 25 mg / L; the concentration of Rhus chinensis leaf extract in promoter group 3 was 50 μg / L, and the concentration of sodium selenite was 50 mg / L), and cultured for another 5 days. Haematococcus pluvialis from the control group and promoter groups 1 to 3 were centrifuged and the supernatant culture medium was quantitatively analyzed using ICP-MS (inductively coupled plasma mass spectrometry) to determine the sodium selenite content in the culture medium and thus determine the conversion efficiency of inorganic selenium by the algae. The results are shown in Table 1. Scanning electron microscopy was then used to observe the internal morphology of the algae cells. Nano-selenium particles were observed in promoter groups 1 to 3, but not in the control group.

[0053] Table 1

[0054] control group Accelerator Group 1 Accelerator Group 2 Accelerator Group 3 Selenium conversion efficiency (%) 24％ 85％ 94％ 80％

[0055] The experimental data from the above examples demonstrate that flavonoids can significantly promote nano-selenium production in microalgae. Compared to the nano-selenium content in normally cultured microalgae, nano-selenium production in microalgae treated with flavonoids was significantly increased. The results indicate that flavonoids place the microalgae in a state of stress. After treatment, the microalgae cells appear red, have specialized cell walls, and are filled with lipid droplets. This suggests that flavonoids not only promote nano-selenium production in microalgae, but also promote the accumulation of high-value-added substances such as lipids and astaxanthin.

[0056] In summary, the present application discloses that flavonoids have the effect of promoting the synthesis of nano-selenium by microalgae. The present application provides a promoter and method for promoting the synthesis of nano-selenium by microalgae. The promoter has simple ingredients, including flavonoids, sodium selenite and a solvent. The method comprises: step 1) obtaining microalgae in the plateau phase, mixing the flavonoids with the plateau phase microalgae to obtain treated microalgae; step 2) collecting the treated microalgae in step 1), separating and extracting the nano-selenium in the microalgae. This production method can significantly promote the production of nano-selenium by microalgae, and the method is highly efficient, low-cost, and has broad industrial application prospects.

[0057] The above is only a preferred embodiment of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A method for promoting the synthesis of nano-selenium by microalgae, characterized in that: The method also promotes the accumulation of high value-added substance astaxanthin in microalgae, comprising the following steps: Step 1: obtaining microalgae in a stationary phase, and mixing a promoter with a culture medium containing the microalgae to obtain treated microalgae; wherein the promoter comprises a flavonoid compound, sodium selenite, and a solvent, polyethylene glycol, and the flavonoid compound is quercetin; Step 2, extracting nano-selenium from the treated microalgae; The microalgae is Haematococcus pluvialis; The flavonoid compound is added to the culture medium in an amount of 12.5 to 50 μg / L; The sodium selenite is added to the culture medium in an amount of 0.5-50 mg / L.

2. The method according to claim 1, characterized in that In step 1, the volume percentage of the flavonoid compound in the culture medium is .

3. The method according to claim 1, characterized in that In step 1, the mixed culture time is 1 to 7 days.

4. The method according to claim 1, wherein In step 1, the density of the microalgae in the plateau phase is 1×10 6 -2×10 6 pieces / mL.

5. The method according to claim 1, wherein In step 2, the extraction specifically includes: separating and collecting microalgae from the culture medium of step 1, and then extracting nano-selenium from the microalgae.

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