A method for extracting organic selenium from selenium-rich vegetable milk vetch

Abstract

This invention belongs to the field of organic selenium extraction technology and specifically discloses a method for extracting organic selenium from selenium-rich edible milk vetch. The method comprises the following steps: 1) soil preparation; 2) planting and harvesting the edible milk vetch; 3) sample processing; and 4) extraction and separation of organic selenium. The defatted milk vetch powder obtained in step 3) is weighed, added to a certain volume of sodium hydroxide solution, stirred and extracted at room temperature, and centrifuged. The extract is then dialyzed to obtain an organic selenium extract of the edible milk vetch. In practice, the optimal process conditions for extracting organic selenium, considering the significant energy and raw material savings and better suitability for practical production, are as follows: a sodium hydroxide solution concentration of 0.15 mol / L, a sodium hydroxide solution volume 20 times the sample volume, an extraction time of 2 hours, and two extractions. This method utilizes the high protein content of edible milk vetch during the budding stage, with the protein content of dry samples reaching 26.6% during this stage. Furthermore, the soil selenium content is increased before planting, thereby improving the yield of organic selenium.

Description

Technical Field

[0001] The invention belongs to the technical field of organic selenium extraction, and more specifically relates to a method for extracting organic selenium from selenium-rich vegetable milk vetch. Background Art

[0002] Selenium is an essential trace nutrient for humans and animals. It promotes normal growth and development in both humans and animals and has numerous physiological functions. Selenium enhances the immune system, eliminates free radicals, protects DNA in cells from harmful substances, converts harmful substances into inactive compounds, inhibits and delays cell malignancy, and thus has anti-cancer effects. Selenium also counteracts the toxicity of heavy metals such as mercury, arsenic, cadmium, and thallium. Existing research indicates that over 40 diseases are closely related to selenium nutrition. Selenium deficiency can contribute to aging, cardiovascular disease, Keshan disease, diabetes, osteoarthritis, liver disease, and cancer.

[0003] Existing research shows that plants growing in high-selenium areas can not only enrich selenium in the environment, but also convert inorganic selenium into organic selenium through their own assimilation, forming selenium-supplementing raw materials with high production utilization, no toxic side effects, and scientifically controlled dosage. Therefore, strengthening the research on the utilization of plant selenium resources in high-selenium areas and using them as raw materials to develop various types of selenium-rich products such as medicine, health care, and food with high selenium content and bioavailability is not only a need of the health industry, but also the best way to promote local economic development.

[0004] Milk vetch is a biennial herbaceous plant of the genus Astragalus in the Leguminosae family. It can be used as a vegetable, animal feed, or green manure. Milk vetch is rich in protein. Studies have shown that the protein content of edible milk vetch is higher than that of mung bean sprouts and water spinach, and slightly lower than that of pea seedlings (Qiu Xiaoxuan et al. Analysis of yield, nutritional quality, and hygienic quality of young shoots of edible milk vetch varieties. Journal of Fujian Agricultural Sciences, 2012, 27(6):626-629.). It has also been reported that the protein content of dry samples of edible milk vetch before the budding stage reaches 26.6%. Other studies have shown that milk vetch has a strong ability to enrich and transform selenium in the soil. The selenium content of dry samples of the aboveground parts of milk vetch cultivated with inorganic selenium fertilizer in the soil is as high as 215 mg / kg. To date, there has been no report on the extraction of organic selenium from selenium-enriched edible milk vetch. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a method for extracting organic selenium from selenium-rich edible milk vetch. This method involves two stages: first, cultivating the selenium-rich edible milk vetch to convert inorganic selenium in the soil into organic selenium within the edible milk vetch; and second, extracting the organic selenium from the edible milk vetch using a solvent.

[0006] To achieve the above object, the present invention provides a method for extracting organic selenium from selenium-rich vegetable milk vetch, comprising the following steps:

[0007] Step 1) Soil preparation: After removing stones and plant roots from the collected yellow-brown soil, the soil is air-dried, ground, and passed through a 10-mesh sieve. The sieved soil is stored for later use.

[0008] The yellow-brown soil is the topsoil (0-20 cm) taken from Mufu Village, Mufu Office, Enshi City, Hubei Province, and the soil selenium content is 9.61±0.24 mg / kg.

[0009] Step 2) Planting and harvesting of edible milk vetch: Weigh the soil obtained in step 1) as needed, add sodium selenite (Na2SeO3) and a certain amount of diammonium phosphate into it according to a certain ratio, stir evenly and place in a plastic pot, sow edible milk vetch seeds; harvest the above-ground part when the edible milk vetch grows to the budding stage.

[0010] Step 3) Sample processing: The above-ground part of the vegetable milk vetch harvested in step 2) is air-dried under natural conditions, then dried in an oven at 90° C., crushed until all particles pass through a 100-mesh sieve, degreased with an appropriate amount of acetone, and then blown dry with hot air for later use.

[0011] Step 4), extraction and separation of organic selenium: weigh the defatted milk vetch powder obtained in step 3), add a certain volume of sodium hydroxide solution, stir and extract at room temperature, and centrifuge; the extract is placed in a dialysis bag for dialysis to obtain an organic selenium extract of edible milk vetch, which is further dried and crushed to obtain an organic selenium solid powder.

[0012] Furthermore, in step 2), the on-demand weighing refers to a weighed mass of 5 to 15 kg, preferably 7 to 13 kg, and more preferably 10 kg; the sodium selenite is added according to a certain ratio (the ratio refers to the mass ratio of sodium selenite to soil in mg / kg), and the certain ratio is 5 to 15 mg / kg, preferably 8 to 12 mg / kg, and more preferably 10 mg / kg; the certain amount of diammonium hydrogen phosphate is added, and the certain amount is 3 to 9 g per 10 kg of soil, preferably 4 to 8 g, and more preferably 6 g.

[0013] Furthermore, the concentration of the sodium hydroxide solution is 0.05 to 0.25 mol / L, preferably 0.10 to 0.2 mol / L, and more preferably 0.15 mol / L.

[0014] Furthermore, the volume of the sodium hydroxide solution is 10 to 30 times the volume of the weighed defatted Chinese milk vetch powder (the volume of the weighed defatted Chinese milk vetch powder is measured by placing it into a measuring cylinder), preferably 15 to 25 times, and more preferably 20 times.

[0015] Furthermore, the extraction time of the stirring extraction at room temperature is 0.5 to 2.5 hours, preferably 1.5 to 2.5 hours, and more preferably 2 hours.

[0016] Furthermore, the extraction times of stirring extraction at room temperature is 1 to 4 times, preferably 1 to 3 times, more preferably 2 times.

[0017] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0018] (1) Vegetable milk vetch is a daily edible plant that is easy to plant and harvest. The present invention selects vegetable milk vetch at the budding stage, which can not only ensure the yield of vegetable milk vetch, but also improve the yield of organic selenium because the protein content in the dry sample of vegetable milk vetch at the budding stage reaches a high level of 26.6% (Lan Zhongming et al. Analysis of nutritional components of tender stems and leaves of vegetable milk vetch "Min Zi No. 7". Fujian Agricultural Science and Technology, 2012, 3:108-110.).

[0019] (2) Before planting milk vetch, add sodium selenite (Na2SeO3) to the soil at a ratio of 10 mg / kg to increase the selenium content in the soil. Make full use of the strong enrichment and conversion ability of edible milk vetch on selenium in the soil, convert as much inorganic selenium as possible into organic selenium, and increase the organic selenium content in edible milk vetch.

[0020] (3) By analyzing the occurrence forms of selenium in edible milk vetch, it was found that the main form of organic selenium is protein selenium. The mass fraction of organic selenium in edible milk vetch accounts for 92.49% of the total selenium, of which the content of selenium in alkali-soluble protein accounts for 69.45% of the total selenium.

[0021] (4) The results showed that the total selenium content in alkali-soluble protein and alkali-soluble polysaccharide extracted from astragalus with sodium hydroxide solution as solvent accounted for more than 70% of the total selenium content and more than 76% of the organic selenium content. This indicates that most of the organic selenium in astragalus can be extracted when sodium hydroxide solution is used as the extraction solvent. The results showed that the total selenium content in astragalus with sodium hydroxide solution as the extraction solvent was more than 70% of the total selenium content and more than 76% of the organic selenium content. 4 ) Orthogonal experiments were conducted to determine the influence of various factors on the yield of organic selenium (and, from the perspective of improving the yield of organic selenium in asparagus and saving raw materials and time), the optimal process conditions were determined: the concentration of sodium hydroxide solution was 0.15 mol / L, the amount of sodium hydroxide solution was 20 times the sample volume, the extraction time was 2 hours, and the number of extractions was 2 times (this combination can save a lot of energy and raw materials). DETAILED DESCRIPTION

[0022] The applicant will now further explain the present invention in detail with reference to examples conducted at the Experimental Engineering Center of Hubei University for Nationalities in Enshi, Hubei Province, with the aim of enabling those skilled in the art to have a more detailed understanding and knowledge of the present invention. The following examples should not be construed as limiting the scope of protection claimed in the present invention to any extent.

[0023] Test materials

[0024] The experimental materials (vegetable milk vetch, variety: Min Zi No. 7, the same below, no further details) came from soil pots. The soil samples were yellow-brown soil (topsoil, 0-20 cm) and were taken from Mufu Village, Mufu Office, Enshi City, Hubei Province. After removing stones and plant rhizomes, the collected soil samples were air-dried, ground, sieved, and stored for later use. The selenium content in the soil in the area was 9.61±0.24 mg / kg. Before sowing vegetative milk vetch, 10 mg / kg of sodium selenite (Na2SeO3) was added to 10 kg of treated soil, and 6 g of diammonium hydrogen phosphate was added. The experiment was carried out at the Experimental Engineering Center of Hubei University for Nationalities in Enshi, Hubei. The sowing time was October 2021. It was routinely managed under natural conditions and harvested in April 2022 during the budding period of vegetative milk vetch.

[0025] Main test instruments

[0026] HGF-T atomic fluorescence spectrometer (China); AVANTI30 high-speed refrigerated centrifuge (Beckman Co., USA); ETHOS PLUS microwave digestion system (Italy); S53 / 54 full-wavelength spectrophotometer (Shanghai Lingguang Scientific Instrument Factory).

[0027] Determination method

[0028] The protein content was determined by the Coomassie brilliant blue method, and the standard curve was prepared using bovine serum albumin. The linear regression equation between the absorbance of the protein and its concentration was Y = 432.18x - 0.325 (R 2 =0.9943), where Y represents the absorbance of the protein and x represents the concentration of the protein. When measuring the sample, the absorbance of the protein is first measured, and then the absorbance is substituted into the above regression equation to calculate the protein concentration.

[0029] The content of soluble polysaccharide was determined by phenol-sulfuric acid colorimetry, and the standard curve was prepared with glucose. The linear regression equation between the absorbance of polysaccharide and its concentration was Y = 123.58x + 1.325 (R 2 =0.9929), where Y represents the absorbance of glucose and x represents the concentration of glucose. When measuring the sample, the absorbance of the polysaccharide is first measured, and then the absorbance is substituted into the above regression equation to calculate the concentration of the polysaccharide.

[0030] The content of selenium was determined by atomic fluorescence spectrometry. After digestion, selenium powder was prepared into a series of standard solutions to make a standard curve. The linear regression equation between the fluorescence intensity of selenium and its concentration was Y = 0.0238x - 0.0192 (R 2 =0.9913), where Y represents the fluorescence intensity of selenium and x represents the concentration of selenium. When measuring a sample, the fluorescence intensity of selenium is first measured, and then the absorbance is substituted into the above regression equation to calculate the concentration of selenium.

[0031] Test methods

[0032] 1. Sample Processing

[0033] After harvesting, the above-ground parts of the edible milk vetch are air-dried under natural conditions, then dried in an oven at 90°C, crushed until all particles pass through a 100-mesh sieve, de-esterified with an appropriate amount of acetone, and blown dry with hot air for later use.

[0034] 2. Separation of organic matter from Chinese milk vetch

[0035] Weigh 3 portions of 1.00 g of defatted astragalus powder in parallel, add 20 times the volume of water to each of them (the volume here is the measured volume of the defatted vegetable astragalus powder, and the measured volume is the volume obtained by measuring the defatted vegetable astragalus powder in a measuring cylinder. The same applies hereinafter, and will not be repeated here.), stir in an 80°C water bath for 2 hours, and put into a dialysis bag (molecular weight cut-off of 10,000 Daltons. The same applies hereinafter, and will not be repeated here) for dialysis to remove small molecules. The remaining portion in the dialysis bag is approximately regarded as the organic substance of astragalus.

[0036] 3. Extraction and separation of protein from Chinese milk vetch

[0037] Weigh 1.00 g of deesterified Chinese milk vetch powder from four portions in parallel and add 20 volumes of water, 0.15 mol / L sodium chloride solution, 0.15 mol / L hydrochloric acid solution, and 0.15 mol / L sodium hydroxide solution, respectively. Stir and extract at room temperature for 2 hours, then centrifuge. Repeat the extraction twice, and combine the three extracts to obtain water-soluble protein extract, salt-soluble protein extract, acid-soluble protein extract, and alkali-soluble protein extract of Chinese milk vetch, respectively.

[0038] Add ammonium sulfate to the extracts of water-soluble protein, salt-soluble protein, acid-soluble protein and alkali-soluble protein respectively until saturated, let stand at 4°C overnight, centrifuge, and dialyze the precipitate into the dialysate. When no precipitate is formed after adding saturated barium chloride, fully dissolve it with distilled water, 0.15mol / L sodium chloride, 0.15mol / L hydrochloric acid and 0.15mol / L sodium hydroxide solution, and then make up to 100mL for use.

[0039] 4. Extraction and separation of soluble polysaccharides from Chinese milk vetch

[0040] Weigh 1.00 g of deesterified Chinese milk vetch powder from three portions in parallel, add 25 volumes of water, 0.1 mol / L hydrochloric acid solution, and 0.1 mol / L sodium hydroxide solution, respectively. Stir and extract in a boiling water bath for 1 hour, then centrifuge. Repeat the extraction twice, and combine the three extracts to obtain a water-soluble polysaccharide extract, an acid-soluble polysaccharide extract, and an alkali-soluble polysaccharide extract.

[0041] Add equal volumes of Sevage reagent to the water-soluble, acid-soluble, and alkali-soluble polysaccharide extracts, shake, and allow to stand for separation, repeat five times, and then perform alcohol precipitation twice. Dissolve the extracts thoroughly with distilled water, 0.1 mol / L hydrochloric acid, and 0.1 mol / L sodium hydroxide solution, respectively, and dilute to 50 mL for later use.

[0042] 5. Extraction and separation of organic selenium from Chinese milk vetch

[0043] Weigh 1.00g of deesterified milk vetch powder and add 20 times the volume of 0.15mol / L sodium hydroxide solution. Stir and extract at room temperature for 2 hours, then centrifuge. The extract is dialyzed to obtain a milk vetch macromolecular organic selenium extract, which can be regarded as the selenium-containing organic matter extracted from milk vetch.

[0044] 6. Digestion

[0045] Take a certain amount of deesterified astragalus powder, astragalus organic matter, various proteins, soluble polysaccharides and selenium-containing organic matter extracted from astragalus and put them into a digestion tank, add 7mL of concentrated HNO3 (68wt%) and 2mL of 30% H2O2, digest in a microwave digestion system, cool and adjust the volume to 100mL for use.

[0046] 7. Calculation of organic selenium yield

[0047] The organic selenium yield η=(m1 / m)×100%, wherein m1 is the mass of organic selenium extracted from the Chinese milk vetch powder, and m is the total mass of selenium in the Chinese milk vetch powder.

[0048] The occurrence forms of selenium in Chinese milk vetch

[0049] 1. Distribution of organic and inorganic selenium in astragalus

[0050] Based on the above experiments, the organic matter in milk vetch was separated, and the mass fractions of organic and total selenium in the milk vetch were determined. The mass fraction of inorganic selenium was also calculated, and the results are shown in Table 1. The results showed that selenium in milk vetch exists primarily in an organically bound form, accounting for 92.49% of the total selenium in the milk vetch.

[0051] Table 1 Distribution of selenium occurrence forms in Chinese milk vetch

[0052]

[0053] 2. Selenium content in the main organic components of Chinese milk vetch

[0054] Before the budding stage, the main organic components in vegetable milk vetch are protein and soluble polysaccharides. In order to further study the distribution of selenium in the main organic components, according to the above experiment, various different proteins (water-soluble protein, salt-soluble protein, acid-soluble protein and alkali-soluble protein) and various different polysaccharides (water-soluble polysaccharides, acid-soluble polysaccharides and alkali-soluble polysaccharides) were extracted from vegetable milk vetch respectively, and the selenium content in each organic component was then measured. The results are shown in Table 2. As shown in Table 2, the content of alkali-soluble protein selenium is the highest among the various protein seleniums, while the content of selenium in acid-soluble protein and water-soluble protein is relatively low, and the content of selenium in alkali-soluble protein accounts for 69.45% of the total selenium. In addition, the content of selenium in various polysaccharides is low, and among the three different soluble polysaccharides, the content of selenium in alkali-soluble polysaccharides is relatively high.

[0055] Table 2 Distribution of selenium in the main components of edible milk vetch

[0056]

[0057] Single-factor experiment

[0058] The above test results show that the sum of the selenium content in the alkali-soluble proteins and alkali-soluble polysaccharides extracted from Chinese milk vetch using alkali as the solvent accounts for over 70% of the total selenium and over 76% of the organic selenium. This indicates that using alkali as the extraction solvent can extract the majority of the organic selenium in edible Chinese milk vetch. Considering cost, sodium hydroxide solution was selected as the extraction solvent for organic selenium in edible Chinese milk vetch in this experiment.

[0059] Single Factor Experiment on Extraction of Organic Selenium from Vegetable Milkvetch

[0060] The concentration of sodium hydroxide solution, the amount of sodium hydroxide solution, the extraction time and the number of extractions were used as factors affecting the yield of organic selenium. Different levels of each factor were set to find out the influence of different factors on the selenium yield.

[0061] 1. Effect of sodium hydroxide solution concentration (A) on the extraction of organic selenium

[0062] Different concentrations of sodium hydroxide solution were used as the extraction solvent to extract organic selenium from milk vetch samples. Five sodium hydroxide concentration gradients were set up in this experiment: 0.05 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, and 0.25 mol / L. Other extraction conditions included using 20 volumes of sodium hydroxide solution at room temperature. Each sample was extracted twice, with each extraction lasting 2 hours. The results are shown in Table 3. As shown in Table 3, the yield of organic selenium in milk vetch increased with increasing sodium hydroxide concentration within the range of 0.05 to 0.15 mol / L. Thereafter, the yield gradually decreased. This is primarily due to the denaturation of some proteins and reduced solubility when the sodium hydroxide concentration is too high. Therefore, the optimal sodium hydroxide concentration for extracting organic selenium from milk vetch is 0.15 mol / L.

[0063] Table 3 Effect of sodium hydroxide solution concentration (A) on organic selenium extraction effect

[0064]

[0065] 2. Effect of sodium hydroxide solution dosage (B) on organic selenium extraction

[0066] To investigate the effect of sodium hydroxide solution dosage on the extraction of organic selenium from Chinese milk vetch, this experiment used different sodium hydroxide solution dosages to extract organic selenium from samples. Five different sodium hydroxide solution dosages were used: 10, 15, 20, 25, and 30 times the sample volume. Extraction conditions included 0.15 mol / L sodium hydroxide solution at room temperature. Each sample was extracted twice, each for 2 hours. The results are shown in Table 4. As shown in Table 4, the yield of organic selenium from Chinese milk vetch increased with increasing sodium hydroxide dosage, and the yield leveled off after the dosage reached 20 times the sample volume. To conserve experimental materials, a sodium hydroxide dosage of 20 times the sample volume is appropriate.

[0067] Table 4 Effect of sodium hydroxide solution dosage (B) on organic selenium extraction effect

[0068]

[0069] 3. Effect of extraction time (C) on extraction effect

[0070] Extraction time is a key factor influencing the yield of organic selenium. To investigate the effect of extraction time on the extraction of organic selenium from Chinese milk vetch, this experiment employed five different extraction times: 0.5, 1.0, 1.5, 2.0, and 2.5 hours per extraction. The remaining extraction conditions involved using 20 times the volume of 0.15 mol / L sodium hydroxide solution at room temperature, with each sample extracted twice. The results are shown in Table 5. As shown in Table 5, the yield of organic selenium in Chinese milk vetch increases with extraction time, with the yield increasing minimally when the extraction time exceeds 2.0 hours. Considering time and energy consumption, an extraction time of 2.0 hours per extraction is optimal.

[0071] Table 5 Effect of extraction time (C) on the extraction effect of organic selenium

[0072]

[0073] 4. Effect of extraction times (D) on extraction effect

[0074] The number of extractions is another important factor influencing the yield of organic selenium. To examine the effect of the number of extractions on the extraction efficiency of organic selenium from Chinese milk vetch, this experiment set up four different extraction times: one, two, three, and four extractions. The other extraction conditions were 20 times the volume of 0.15 mol / L sodium hydroxide solution at room temperature, and each sample was extracted for two hours. The results are shown in Table 6. As shown in Table 6, the yield of organic selenium in Chinese milk vetch increases with the number of extractions. When the extraction time exceeds two times, the yield of organic selenium increases less. Considering the time and energy consumption, two extractions per sample is appropriate.

[0075] Table 6 Effect of extraction times (D) on the extraction effect of organic selenium

[0076]

[0077] Orthogonal test on the extraction of organic selenium from Chinese milk vetch

[0078] With the results of the single factor test as a reference, a 4-factor 3-level orthogonal test was conducted on the four factors of sodium hydroxide solution concentration, sodium hydroxide solution dosage, extraction time and extraction times. The orthogonal design table of factor levels is shown in Table 7. Taking the organic selenium yield in milk vetch as an indicator, the L9(3 4 ) Orthogonal experiment was used to determine the optimal extraction process conditions.

[0079] Table 7 Orthogonal design factor level table

[0080]

[0081] Orthogonal experiment

[0082] Using L9(34 ) Orthogonal tables were used to extract organic selenium from milk vetch samples. The yield of organic selenium was used as an indicator to determine the optimal extraction conditions for organic selenium from milk vetch under the combined effects of various factors. The results and variance analysis are shown in Tables 8 and 9.

[0083] Table 8 Orthogonal test table and test results for organic selenium extraction from milk vetch powder

[0084]

[0085]

[0086] Table 9 Variance analysis of orthogonal test for extraction of organic selenium from milk vetch powder

[0087]

[0088] Note: * indicates significant level

[0089] The test results in Table 8 indicate that the factors affecting the yield of organic selenium from edible milk vetch are, in the following order: number of extractions > amount of sodium hydroxide solution > concentration of sodium hydroxide solution > extraction time. The variance analysis in Table 9 shows that the number of extractions significantly affects the yield of organic selenium from edible milk vetch powder, while other factors do not significantly affect the yield.

[0090] The test results in Table 8 and the variance analysis in Table 9 show that the A1B3C3D3 combination had the highest organic selenium yield. Neither the sodium hydroxide solution dosage (B) nor the extraction time (C) had a significant effect on the organic selenium yield. However, a single-factor experiment revealed that increasing the sodium hydroxide solution concentration from 0.10 mol / L to 0.15 mol / L significantly increased the organic selenium yield. Furthermore, while the number of extractions significantly affected the organic selenium yield, a single-factor experiment revealed that increasing the number of extractions from 2 to 3 did not significantly increase the yield.

[0091] Example 1

[0092] The method for extracting organic selenium from selenium-rich vegetable milk vetch comprises the following steps:

[0093] Step 1) Soil preparation: After removing stones and plant roots from the collected yellow-brown soil, the soil is air-dried, ground, and passed through a 10-mesh sieve. The sieved soil is stored for later use.

[0094] The yellow-brown soil is the topsoil (0-20 cm) taken from Mufu Village, Mufu Office, Enshi City, Hubei Province, and the soil selenium content is 9.61±0.24 mg / kg.

[0095] Step 2) Planting and harvesting of vegetable milk vetch: Weigh 10 kg of the soil obtained in step 1), add sodium selenite (Na2SeO3) at a ratio of 10 mg / kg, and add 6 g of diammonium hydrogen phosphate, stir evenly, and place in a plastic pot, sow vegetable milk vetch seeds; harvest the above-ground part when the vegetable milk vetch grows to the budding stage.

[0096] Step 3) Sample processing: The above-ground part of the vegetable milk vetch harvested in step 2) is air-dried under natural conditions, then dried in an oven at 90° C., crushed until all particles pass through a 100-mesh sieve, degreased with an appropriate amount of acetone, and then blown dry with hot air for later use.

[0097] Step 4), extraction and separation of organic selenium: weigh the defatted vegetable astragalus powder obtained in step 3), add 20 times the volume of 0.10 mol / L sodium hydroxide solution based on the volume obtained by measuring the defatted vegetable astragalus powder in a measuring cylinder, stir and extract at room temperature for 2 hours, and centrifuge; extract twice in total, collect the extract, put it into a dialysis bag for dialysis, and obtain an organic selenium extract of vegetable astragalus by dialysis, which is further dried and crushed to obtain an organic selenium solid powder.

[0098] Example 2

[0099] The method for extracting organic selenium from selenium-rich vegetable milk vetch comprises the following steps:

[0100] Step 1) Soil preparation: After removing stones and plant roots from the collected yellow-brown soil, the soil is air-dried, ground, and passed through a 10-mesh sieve. The sieved soil is stored for later use.

[0101] The yellow-brown soil is the topsoil (0-20 cm) taken from Mufu Village, Mufu Office, Enshi City, Hubei Province, and the soil selenium content is 9.61±0.24 mg / kg.

[0102] Step 2) Planting and harvesting of vegetable milk vetch: Weigh 10 kg of the soil obtained in step 1), add sodium selenite (Na2SeO3) at a ratio of 10 mg / kg, and add 6 g of diammonium hydrogen phosphate. Stir well and place in a plastic pot, sow vegetable milk vetch seeds; harvest the above-ground part when the vegetable milk vetch grows to the budding stage.

[0103] Step 3) Sample processing: The above-ground part of the vegetable milk vetch harvested in step 2) is air-dried under natural conditions, then dried in an oven at 90° C., crushed until all particles pass through a 100-mesh sieve, degreased with an appropriate amount of acetone, and then blown dry with hot air for later use.

[0104] Step 4), extraction and separation of organic selenium: weigh the defatted vegetable astragalus powder obtained in step 3), add 20 times the volume of 0.15 mol / L sodium hydroxide solution based on the volume obtained by measuring the defatted vegetable astragalus powder in a measuring cylinder, stir and extract at room temperature for 2 hours, and centrifuge; extract twice in total, collect the extract and put it into a dialysis bag for dialysis, obtain an organic selenium extract of vegetable astragalus by dialysis, further dry and crush to obtain an organic selenium solid powder.

[0105] Example 3

[0106] The method for extracting organic selenium from selenium-rich vegetable milk vetch comprises the following steps:

[0107] Step 1) Soil preparation: After removing stones and plant roots from the collected yellow-brown soil, the soil is air-dried, ground, and passed through a 10-mesh sieve. The sieved soil is stored for later use.

[0108] The yellow-brown soil is the topsoil (0-20 cm) taken from Mufu Village, Mufu Office, Enshi City, Hubei Province, and the soil selenium content is 9.61±0.24 mg / kg.

[0109] Step 2) Planting and harvesting of vegetable milk vetch: Weigh 10 kg of the soil obtained in step 1), add sodium selenite (Na2SeO3) at a ratio of 10 mg / kg, and add 6 g of diammonium hydrogen phosphate. Stir well and place in a plastic pot, sow vegetable milk vetch seeds; harvest the above-ground part when the vegetable milk vetch grows to the budding stage.

[0110] Step 3) Sample processing: The above-ground part of the vegetable milk vetch harvested in step 2) is air-dried under natural conditions, then dried in an oven at 90° C., crushed until all particles pass through a 100-mesh sieve, degreased with an appropriate amount of acetone, and then blown dry with hot air for later use.

[0111] Step 4), extraction and separation of organic selenium: weigh the defatted vegetable astragalus powder obtained in step 3), add 20 times the volume of 0.20 mol / L sodium hydroxide solution based on the volume obtained by measuring the defatted vegetable astragalus powder in a measuring cylinder, stir and extract at room temperature for 2 hours, and centrifuge; extract twice in total, collect the extract, put it into a dialysis bag for dialysis, and obtain an organic selenium extract of vegetable astragalus by dialysis, which is further dried and crushed to obtain an organic selenium solid powder.

[0112] Example 4

[0113] The method for extracting organic selenium from selenium-rich vegetable milk vetch comprises the following steps:

[0114] Step 1) Soil preparation: After removing stones and plant roots from the collected yellow-brown soil, the soil is air-dried, ground, and passed through a 10-mesh sieve. The sieved soil is stored for later use.

[0115] The yellow-brown soil is the topsoil (0-20 cm) taken from Mufu Village, Mufu Office, Enshi City, Hubei Province, and the soil selenium content is 9.61±0.24 mg / kg.

[0116] Step 2) Planting and harvesting of vegetable milk vetch: Weigh 10 kg of the soil obtained in step 1), add sodium selenite (Na2SeO3) at a ratio of 10 mg / kg, and add 6 g of diammonium hydrogen phosphate, stir evenly and place in a flowerpot, sow vegetable milk vetch seeds; harvest the above-ground part when the vegetable milk vetch grows to the bud stage.

[0117] Step 3) Sample processing: The above-ground part of the vegetable milk vetch harvested in step 2) is air-dried under natural conditions, then dried in an oven at 90° C., crushed until all particles pass through a 100-mesh sieve, degreased with an appropriate amount of acetone, and then blown dry with hot air for later use.

[0118] Step 4), extraction and separation of organic selenium: weigh the defatted vegetable milk vetch powder obtained in step 3), add 20 times the volume of 0.15 mol / L sodium hydroxide solution based on the volume obtained by measuring the weighed defatted vegetable milk vetch powder in a measuring cylinder, stir and extract at room temperature for 2.5 hours, and centrifuge; extract twice in total, collect the extract and put it into a dialysis bag for dialysis, obtain an organic selenium extract of vegetable milk vetch by dialysis, further dry and crush to obtain an organic selenium solid powder.

[0119] Example 5

[0120] The method for extracting organic selenium from selenium-rich vegetable milk vetch comprises the following steps:

[0121] Step 1) Soil preparation: After removing stones and plant roots from the collected yellow-brown soil, the soil is air-dried, ground, and passed through a 10-mesh sieve. The sieved soil is stored for later use.

[0122] The yellow-brown soil is the topsoil (0-20 cm) taken from Mufu Village, Mufu Office, Enshi City, Hubei Province, and the soil selenium content is 9.61±0.24 mg / kg.

[0123] Step 2) Planting and harvesting of vegetable milk vetch: Weigh 10 kg of the soil obtained in step 1), add sodium selenite (Na2SeO3) at a ratio of 10 mg / kg, and add 6 g of diammonium hydrogen phosphate, stir evenly and place in a flowerpot, sow vegetable milk vetch seeds; harvest the above-ground part when the vegetable milk vetch grows to the bud stage.

[0124] Step 3) Sample processing: The above-ground part of the vegetable milk vetch harvested in step 2) is air-dried under natural conditions, then dried in an oven at 90° C., crushed until all particles pass through a 100-mesh sieve, degreased with an appropriate amount of acetone, and then blown dry with hot air for later use.

[0125] Step 4), extraction and separation of organic selenium: weigh the defatted vegetable asparagus powder obtained in step 3), add 25 times the volume of 0.15 mol / L sodium hydroxide solution based on the volume obtained by measuring the defatted vegetable asparagus powder in a measuring cylinder, stir and extract at room temperature for 2 hours, and centrifuge; extract twice in total, collect the extract, put it into a dialysis bag for dialysis, and obtain an organic selenium extract of vegetable asparagus by dialysis, which is further dried and crushed to obtain an organic selenium solid powder.

[0126] Based on the results of Examples 1-5, Example 2 represents the optimal process conditions for increasing the yield of organic selenium in edible Chinese milk vetch while also saving raw materials and time: a sodium hydroxide concentration of 0.15 mol / L, an amount of sodium hydroxide equal to 20 times the sample volume, an extraction time of 2 hours, and two extractions. Under these conditions, the yield of organic selenium in edible Chinese milk vetch was measured to be 68.92%. While slightly lower than the A1B3C3D3 combination in the orthogonal experiment, this combination can save significant energy and raw materials.

[0127] The above-described embodiments merely represent specific implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for extracting organic selenium from selenium-rich vegetable milk vetch, comprising the following steps: Step 1) Soil preparation: After removing stones and plant roots from the collected yellow-brown soil, air-dry it, grind it into powder, and pass it through a 10-mesh sieve. The sieved soil is stored for later use; The yellow-brown soil is the topsoil 0-20 cm below the surface, and the soil selenium content is 9.61±0.24 mg / kg; Step 2), Planting and harvesting of vegetable milk vetch: Weigh the soil obtained in step 1) as needed, add sodium selenite Na2SeO3 according to a certain ratio, and then add a certain amount of diammonium hydrogen phosphate. Stir evenly and place in a plastic pot, and sow vegetable milk vetch seeds; harvest the above-ground part of the vegetable milk vetch when it grows to the budding stage; the variety of the vegetable milk vetch is Min Zi No. 7, the weighing as needed means weighing 10 kg, the certain ratio is 10 mg sodium selenite / kg soil, and the certain amount is 6 g diammonium hydrogen phosphate / 10 kg soil; Step 3) Sample processing: The above-ground part of the Chinese milk vetch harvested in step 2) was air-dried under natural conditions, then dried in an oven at 90°C, pulverized until all particles passed through a 100-mesh sieve, degreased with an appropriate amount of acetone, and then dried with hot air for later use; Step 4), extraction and separation of organic selenium: weigh the defatted vegetable milk vetch powder obtained in step 3), add a certain volume of a 0.15 mol / L sodium hydroxide solution, stir and extract at room temperature, and centrifuge; the extract is placed in a dialysis bag and dialyzed to obtain an organic selenium extract of vegetable milk vetch, which is further dried and crushed to obtain an organic selenium solid powder; The certain volume means that the volume of the sodium hydroxide solution is 20 times the volume of the weighed defatted vegetable milk vetch powder, wherein the volume of the weighed defatted vegetable milk vetch powder is obtained by placing the weighed defatted vegetable milk vetch powder into a measuring cylinder; the extraction time for the stirring extraction at room temperature is 2 hours and the number of extractions is 2 times.

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