Selenium protein with high antioxidant activity as well as preparation method and application of selenium protein

Abstract

The invention relates to the technical field of extraction of microbial functional components, in particular to selenoprotein with high antioxidant activity as well as a preparation method and application of the selenoprotein. The selenoprotein provided by the invention is a selenoprotein substance with more prominent yeast source and high biological activity compared with selenium-enriched yeast, the antioxidant activity of the selenoprotein is 10.08-14.52 times of that of total selenoprotein and 9.14-13.17 times of that of the selenium yeast under the isoconcentration condition, the selenium content is 3498.09-4730.47 mg / kg, and the selenoprotein has the advantages of high activity and efficient absorption compared with the selenium-enriched yeast; wide application prospects are realized in the aspects of medicine, functional foods, cosmetics and the like.

Description

Technical Field

[0001] The invention relates to the technical field of microbial functional component extraction, and in particular to a selenoprotein with high antioxidant activity and a preparation method and application thereof. Background Art

[0002] Selenium is one of the essential trace elements for the human body. It can play an antioxidant and free radical scavenging role in the human body and is known as an antioxidant star. The antioxidant capacity of selenium is 300 to 500 times that of vitamin E. It can also remove heavy metal toxins remaining in the human body. In the general environment of selenium deficiency, reasonable selenium supplementation is very important for human health.

[0003] Compared with traditional sodium selenite selenium supplement preparations, selenium-enriched yeast has the advantages of safety and high absorption rate. It has certain preventive and auxiliary therapeutic effects on gastric cancer, intestinal cancer, lung cancer, liver cancer, breast cancer, malignant lymphoma, and germ cell tumors. The essence of selenium-enriched yeast is that during the growth and proliferation of selenium-enriched yeast, the yeast itself assimilates the selenium source. After the selenium source enters the yeast cell, it combines with the yeast's own amino acids, proteins and polysaccharides to form organic selenoproteins and selenium polysaccharides. Selenium-enriched yeast is a single-cell fungal organic selenium preparation. Its structure contains glucan, mannan, glycoprotein and chitin as cell protection components. It has certain difficulties in digestion and absorption in living organisms, resulting in the inability to better absorb the organic selenium in the yeast. Therefore, extracting organic selenoproteins from selenium-enriched yeast as a selenium supplement can be better digested and absorbed by living organisms.

[0004] For example, Chinese invention patent CN102775466A uses high-pressure homogenization to crush selenium-enriched yeast, then centrifuges to add high-quality ammonium sulfate to the centrifugal supernatant to collect precipitated protein, dissolves the precipitate with buffer, and then puts it into a dialysis bag for dialysis, dehydrates and concentrates it to a smaller volume with polyethylene glycol, and vacuum freeze-dries it to obtain crude protein. The crude protein powder is dissolved in a small amount of buffer, passed through a microporous filter membrane, and then placed on a dextran gel column to elute and collect the protein components. The invention technology is prepared by high-pressure homogenization and wall breaking in the selenoprotein preparation process, but a large amount of ammonium sulfate needs to be added during the purification process to enrich the selenoprotein, and then desalination is performed by dialysis, which is time-consuming and cumbersome, and is not conducive to large-scale industrial production.

[0005] At present, according to the research results of existing selenium-enriched yeast protein extraction and preparation methods, although a variety of extraction and preparation methods have been reported, there are few reports on the extraction of yeast selenoproteins with high antioxidant activity. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a selenoprotein with high antioxidant activity and a preparation method and application thereof. The present invention provides a selenoprotein with high antioxidant activity, which has higher antioxidant activity than selenium-enriched yeast and has broad application prospects in medicine, functional foods and cosmetics.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a selenoprotein with high antioxidant activity, characterized in that the selenoprotein with high antioxidant activity has a molecular weight of 36.476-42.117 KD and a selenium content of 3498.09-4730.47 mg / kg.

[0009] Preferably, the selenoprotein with high antioxidant activity is composed of the following amino acids in molar percentage: selenomethionine 3.47% to 7.21%, aspartic acid 10.10 to 15.34%, threonine 6.73 to 8.49%, serine 26.13 to 28.27%, glutamate 2.37 to 5.39%, glycine 6.41 to 8.72%, alanine 7.33 to 15.94%, valine 2.85 to 4.76%, lysine 4.31 to 5.59%, histidine 5.18 to 7.93%, and selenocysteine ​​6.54 to 9.62%.

[0010] Preferably, the selenoprotein with the highest antioxidant activity is composed of the following amino acids in molar percentage: selenomethionine 5.21%, aspartic acid 10.10%, threonine 8.49%, serine 28.27%, glutamate 5.39%, glycine 8.70%, alanine 8.94%, valine 3.76%, lysine 5.59%, histidine 7.93% and selenocysteine ​​7.62%.

[0011] The present invention provides a method for preparing the selenoprotein with high antioxidant activity described in the above technical solution, comprising the following steps:

[0012] Mixing selenium-enriched yeast and water to obtain a selenium-enriched yeast suspension;

[0013] Extracting selenoproteins from the selenium-enriched yeast suspension to obtain a selenoprotein extract; the extraction method includes: high-pressure homogenization extraction and / or high-static pressure extraction;

[0014] Adjusting the pH value of the selenoprotein extract to acidic to perform protein precipitation to obtain a precipitate;

[0015] The precipitate is separated by a dextran gel chromatography column, eluted with a phosphate buffer solution of pH=10, and an eluate having an elution volume multiple of 0.15 to 0.67 times the gel filling volume of the dextran gel chromatography column is collected, wherein the eluate contains the selenoprotein with high antioxidant activity.

[0016] Preferably, during the high-pressure homogenization extraction, the homogenization pressure is 800 to 1500 bar, and the number of homogenization times is 1 to 8 times;

[0017] During the high static pressure extraction, the extraction pressure is 50-200 MPa, the number of extractions is 1-3 times, and the time of each extraction is 3-5 minutes.

[0018] Preferably, the extraction method is a combination of high static pressure extraction and high pressure homogenization extraction.

[0019] Preferably, the elution flow rate is 0.5 to 3 mL / min.

[0020] Preferably, the pH value of the selenoprotein extract is adjusted to 3-4.5 for protein precipitation; the temperature of the protein precipitation is 1-4°C.

[0021] Preferably, before extracting the selenium-enriched yeast suspension, the pH value of the selenium-enriched yeast suspension is adjusted to 9.5-12.

[0022] The present invention provides the use of the selenoprotein with high antioxidant activity described in the above technical solution or the yeast selenoprotein prepared by the preparation method described in the above technical solution in selenium supplementation or preparation of a selenium supplement agent.

[0023] Beneficial effects:

[0024] The selenoprotein provided by the present invention is a selenoprotein substance with more outstanding yeast-derived high biological activity than selenium-enriched yeast, the ABTS free radical scavenging rate reaches 55.03-79.27%, the antioxidant activity under equal concentration conditions is 10.08-14.52 times that of total selenoprotein and 9.14-13.17 times that of selenium yeast, the selenium content is 3498.09-4730.47 mg / kg, and compared with selenium-enriched yeast, it has the advantages of strong activity and efficient absorption, and has broad application prospects in medicine, functional food, cosmetics and the like.

[0025] Furthermore, the preparation method provided by the present invention obtains selenoprotein with high antioxidant activity by premixing, extracting, initially separating and finely separating selenium-enriched yeast using a dextran gel chromatography column. The preparation conditions are mild and the selectivity is high, which is suitable for industrial production. The present invention not only improves the application mode of selenium-enriched yeast as a raw material, but also has great significance for exerting the application value of yeast-derived selenoprotein. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0027] Figure 1 A flow chart for preparing the selenoprotein with high antioxidant activity provided by the present invention;

[0028] Figure 2This is a comparison chart of the ABTS free radical scavenging rates of the finely divided selenium-enriched protein in Example 3 and the unfinely divided selenium-enriched total protein and selenium-enriched yeast under the same concentration conditions;

[0029] Figure 3 The amino acid composition analysis results of the selenoprotein in Example 3;

[0030] Figure 4 This is the molecular weight chromatogram of the dextran gel permeation determination of the refined selenoprotein in Example 3. DETAILED DESCRIPTION

[0031] The invention provides a selenoprotein with high antioxidant activity. The ABTS free radical scavenging rate of the selenoprotein with high antioxidant activity is 55.03-79.27%, the molecular weight is 36.476-42.117 KD, and the selenium content is 3498.09-4730.47 mg / kg.

[0032] As an embodiment, the high antioxidant activity selenoprotein is composed of the following molar percentages of amino acids: selenomethionine 3.47% to 7.21%, aspartic acid 10.10 to 15.34%, threonine 6.73 to 8.49%, serine 26.13 to 28.27%, glutamate 2.37 to 5.39%, glycine 6.41 to 8.72%, alanine 7.33 to 15.94%, valine 2.85 to 4.76%, lysine 4.31 to 5.59%, histidine 5.18 to 7.93%, and selenocysteine ​​6.54 to 9.62%.

[0033] As an embodiment, the high antioxidant activity selenoprotein is composed of the following molar percentages of amino acids: selenomethionine 5.21%, aspartic acid 10.10%, threonine 8.49%, serine 28.27%, glutamate 5.39%, glycine 8.70%, alanine 8.94%, valine 3.76%, lysine 5.59%, histidine 7.93% and selenocysteine ​​7.62%.

[0034] The selenoprotein provided by the present invention is a selenoprotein substance with more outstanding yeast-derived high biological activity than selenium-enriched yeast, the ABTS free radical scavenging rate reaches 55.03-79.27%, the antioxidant activity under equal concentration conditions is 10.08-14.52 times that of total selenoprotein and 9.14-13.17 times that of selenium yeast, the selenium content is 3498.09-4730.47 mg / kg, and compared with selenium-enriched yeast, it has the advantages of strong activity and efficient absorption, and has broad application prospects in medicine, functional food, cosmetics and the like.

[0035] The present invention also provides a method for preparing the selenoprotein with high antioxidant activity described in the above technical solution, comprising the following steps:

[0036] Mixing selenium-enriched yeast and water to obtain a selenium-enriched yeast suspension;

[0037] Extracting selenoproteins from the selenium-enriched yeast suspension to obtain a selenoprotein extract; the extraction method includes: high-pressure homogenization extraction and / or high-static pressure extraction;

[0038] Adjusting the pH value of the selenoprotein extract to acidic to perform protein precipitation to obtain a precipitate;

[0039] The precipitate is separated by a dextran gel chromatography column, eluted with a phosphate buffer solution of pH=10, and an eluate having an elution volume multiple of 0.15 to 0.67 times the gel filling volume of the dextran gel chromatography column is collected, wherein the eluate contains the selenoprotein with high antioxidant activity.

[0040] The present invention mixes selenium-enriched yeast and water to obtain a selenium-enriched yeast suspension. As an embodiment, the mass volume ratio of the selenium-enriched yeast and water is 1g:5-20mL; as another embodiment, the mass volume ratio of the selenium-enriched yeast and water can be 1g:5mL, 1g:10mL, 1g:15mL or 1g:20mL.

[0041] After obtaining the selenium-enriched yeast suspension, the present invention extracts selenoproteins in the selenium-enriched yeast suspension to obtain a selenoprotein extract. As an embodiment, before extracting the selenium-enriched yeast suspension, the pH value of the selenium-enriched yeast suspension is adjusted to 9.5-12. As an embodiment, the reagent for adjusting the pH value of the selenium-enriched yeast suspension includes one or more of sodium carbonate, sodium hydroxide and potassium hydroxide. By adjusting the pH value of the selenium-enriched yeast suspension, the present invention can better promote the deprotonation of the selenium-enriched yeast cell wall, promote the full dissolution of yeast protein under alkaline conditions, and improve the preparation efficiency.

[0042] As an embodiment, during the high-pressure homogenization extraction, the homogenization pressure is 800-1500 bar, and the number of homogenizations is 1-8 times. As another embodiment, during the high-pressure homogenization extraction, the homogenization pressure is 1000-1200 bar, and the number of homogenizations is 2-4 times.

[0043] As an embodiment, during the high static pressure extraction, the extraction pressure is 50-200Mpa, the number of extractions is 1-3 times, and the time of each extraction is 3-5 minutes. As another embodiment, during the high static pressure extraction, the extraction pressure is 100-150Mpa, the number of extractions is 2-3 times, and the time of each extraction is 3-4 minutes.

[0044] As an embodiment, the extraction method is to perform high static pressure extraction and high pressure homogenization extraction in sequence. The selenoprotein prepared by the method of high static pressure combined with high pressure homogenization extraction of the present invention has the highest antioxidant activity, an ABTS free radical scavenging rate of 79.27%, and a selenium content of 4730.47 mg / kg.

[0045] As an embodiment, after the extraction is completed, the present invention centrifuges the extract to obtain the selenoprotein extract. As an embodiment, the speed of the centrifugal treatment is 4000-12000 r / min, and the time is 6-20 min. As another embodiment, the speed of the centrifugal treatment is 8000-12000 r / min, and the time is 8-15 min.

[0046] After obtaining the selenoprotein extract, the present invention adjusts the pH value of the selenoprotein extract to acidic to perform protein precipitation to obtain a precipitate. As an embodiment, the pH value of the selenoprotein extract is adjusted to 3-4.5 for protein precipitation; the temperature of the protein precipitation is 1-4°C. As an embodiment, the reagent for adjusting the pH value of the selenoprotein extract includes one or more of phosphoric acid, hydrochloric acid, citric acid and malic acid. As another embodiment, the pH value of the selenoprotein extract is adjusted to 3.8-4.2. The present invention adjusts the pH value of the selenoprotein extract to acidic so that the positive and negative charges on the surface of the selenoprotein are equal, and the selenoprotein aggregates to form a precipitate, thereby initially separating the selenoprotein.

[0047] As an embodiment, the method for separating the precipitate by protein precipitation can be centrifugal treatment. As an embodiment, the rotation speed of the centrifugal treatment is 4000-12000 r / min, and the time is 6-20 min. As another embodiment, the rotation speed of the centrifugal treatment is 8000-12000 r / min, and the time is 8-15 min.

[0048] After obtaining the precipitate, the present invention separates the precipitate through a dextran gel chromatography column, and collects an eluate whose elution volume multiple is 0.15 to 0.67 times the gel filling volume of the dextran gel chromatography column, wherein the eluate contains the selenoprotein with high antioxidant activity. As one embodiment, the flow rate of the elution is 0.5 to 3 mL / min; as another embodiment, the flow rate of the elution is 1 to 2 mL / min. As one embodiment, the dextran gel chromatography column is a dextran gel chromatography column G-100. The present invention obtains selenoprotein with high antioxidant activity by loading the precipitate through a dextran gel chromatography column and performing fine separation with a phosphate buffer eluent.

[0049] As an embodiment, after obtaining the eluate, the eluate is freeze-dried to obtain the selenoprotein with high antioxidant activity.

[0050] The high antioxidant activity selenoprotein obtained by separation through a dextran gel chromatography column is a selenoprotein substance of yeast origin with more prominent high biological activity than selenium-enriched yeast, with an ABTS free radical scavenging rate of 55.03-79.27% ​​and a selenium content of 3498.09-4730.47 mg / kg.

[0051] Based on the above advantages, the present invention provides the use of the yeast selenoprotein described in the above technical solution in selenium supplementation or preparation of a selenium supplement agent.

[0052] In order to further illustrate the present invention, a selenoprotein with high antioxidant activity and its preparation method and application provided by the present invention are described in detail below in combination with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0053] In the following embodiments, Figure 1 The experimental raw material information table in the embodiments or comparative examples is shown in Table 1.

[0054] Table 1 Experimental raw material information table in the examples or comparative examples

[0055] Reagents Purity or type Manufacturer Selenium-enriched yeast Y20190004891 Mudanjiang Lingtai Pharmaceutical Co., Ltd. Dextran Gel G-100 S14034 Shanghai Yuanye Biotechnology Co., Ltd.

[0056] The calculation formulas for the preparation yield and preparation rate of the following examples or comparative examples are as follows:

[0057] Preparation yield = M1 / M2 × 100%;

[0058] Preparation yield = M3 / M1×100%;

[0059] M1=V×A;

[0060] Wherein, M1 is the mass of protein in selenium-enriched yeast protein solution; A is the percentage of protein in selenium-enriched yeast protein solution determined by Kjeldahl nitrogen determination; V is the volume of selenium-enriched yeast protein solution; M2 is the total protein mass in selenium-enriched yeast; and M3 is the precipitated mass of selenium-enriched yeast protein.

[0061] Example 1

[0062] Premixing: Weigh 10g of selenium-enriched yeast, add water according to the ratio of selenium-enriched yeast mass to water volume of 1g:10mL and stir thoroughly. After stirring thoroughly, add sodium hydroxide alkaline reagent dropwise to adjust the system pH to 10 to obtain selenium-enriched yeast suspension.

[0063] Extraction: The selenium-enriched yeast suspension was subjected to high-pressure homogenization extraction, with a homogenization pressure of 800 bar and a homogenization frequency of 4 times. After the extraction, the suspension was centrifuged at a speed of 6000 r / min for 8 minutes to obtain a selenium-enriched yeast protein solution with a preparation yield of 59.64%.

[0064] Initial separation: the selenium-enriched yeast protein solution was fully stirred, phosphoric acid acid reagent was slowly added dropwise, the pH of the system was adjusted to 3.5, and the adjusted selenium-enriched yeast protein solution was placed in a 4°C environment for protein precipitation for 4 hours, and then centrifuged at a speed of 10000 r / min for 8 minutes to obtain yeast total selenoprotein, with a preparation yield of 68.23%.

[0065] Sperm separation: the yeast total selenoprotein is dissolved, loaded on a dextran gel chromatography column, and selectively separated, the eluent is a pH=10 phosphate buffer, the flow rate is 0.5 mL / min, and the eluate whose elution volume is 0.15 to 0.67 times the gel filling volume of the dextran gel chromatography column is collected; the eluate contains the sperm selenoprotein.

[0066] The ABTS method specified in the national standard GB / T 39100-2020 was used to determine the antioxidant activity of the selenoprotein: 0.02 mL of sample solution and 0.18 mL of ABTS solution were taken respectively, mixed evenly, and reacted at room temperature in the dark for 30 minutes. The absorbance of the sample at 734 nm was tested by an ELISA instrument (the sample solvent was adjusted to zero), and the free radical scavenging rate was calculated to be 60.41%. Under the same concentration conditions, the antioxidant activity of the selenoprotein was 11.06 times that of the total selenoprotein in yeast and 10.03 times that of selenium-enriched yeast; according to GB The amino acid determination analysis of method 5009.124 determined that the selenoprotein for selenoprotein consists of selenomethionine, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, lysine, histidine and selenocysteine, with each molar proportion being 3.73%, 13.93%, 6.81%, 27.53%, 5.39%, 6.57%, 14.81%, 2.85%, 4.87%, 6.60% and 6.91%. The molecular weight of the selenoprotein for selenoprotein was 41.513KD as determined by gel permeation chromatography; the selenium content of the selenoprotein for selenoprotein was 3577.89mg / kg as determined by inductively coupled plasma mass spectrometry.

[0067] Example 2

[0068] Premixing: Weigh 10g of selenium-enriched yeast, add water according to the ratio of selenium-enriched yeast mass to water volume of 1g:15mL and stir thoroughly. After stirring thoroughly, add sodium carbonate alkaline reagent dropwise to adjust the system pH to 10 to obtain selenium-enriched yeast suspension.

[0069] Extraction: The selenium-enriched yeast suspension was subjected to high static pressure extraction, the extraction pressure was 100 MPa, each extraction time was 4 min, and the number of repetitions was 2 times. After the extraction, the selenium-enriched yeast protein solution was obtained by centrifugation at a speed of 8000 r / min for 6 min, and the preparation yield was 53.21%.

[0070] Initial separation: the selenium-enriched yeast protein solution was fully stirred, and citric acid was slowly added to adjust the system pH to 4. The adjusted selenium-enriched yeast protein solution was placed in a 4°C environment for protein precipitation for 6 hours, and then centrifuged at 10000 r / min for 8 minutes to obtain total yeast selenoprotein with a preparation yield of 71.47%.

[0071] Sperm separation: the yeast total selenoprotein is dissolved, loaded on a dextran gel chromatography column, and selectively separated, the eluent is a pH=10 phosphate buffer, the flow rate is 3 mL / min, and the eluent whose elution volume is 0.15 to 0.67 times the gel filling volume of the dextran gel chromatography column is collected; the eluent contains the selenoprotein.

[0072] The ABTS method specified in the national standard GB / T 39100-2020 was used to determine the antioxidant activity of the selenoprotein: 0.02 mL of sample solution and 0.18 mL of ABTS solution were taken respectively, mixed evenly, and reacted at room temperature in the dark for 30 minutes. The absorbance value of the sample at 734 nm was tested by an ultraviolet spectrophotometer (the sample solvent was adjusted to zero). The free radical scavenging rate was calculated to be 55.03%. Under the same concentration conditions, the antioxidant activity of the selenoprotein was 10.08 times that of the total selenoprotein of yeast and 9.14 times that of selenium-enriched yeast. The amino acid determination analysis of method 5009.124 determined that the selenoprotein was composed of selenomethionine, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, lysine, histidine and selenocysteine, with each molar proportion being 3.47%, 15.34%, 8.49%, 28.27%, 2.37%, 6.41%, 10.83%, 4.76%, 5.59%, 7.93% and 6.54%. The molecular weight of the selenoprotein was determined to be 42.117KD by gel permeation chromatography; the selenium content of the selenoprotein was determined to be 3498.09mg / kg by inductively coupled plasma mass spectrometry.

[0073] Example 3

[0074] Premixing: Weigh 10g of selenium-enriched yeast, add water according to the ratio of selenium-enriched yeast mass to water volume of 1g:5mL and stir thoroughly. After stirring thoroughly, add sodium hydroxide alkaline reagent dropwise to adjust the system pH to 11 to obtain selenium-enriched yeast suspension.

[0075] Extraction: The selenium-enriched yeast suspension was subjected to high static pressure extraction and high pressure homogenization extraction. First, the extraction pressure was 100 MPa, the extraction time was 3 min, and the number of times was repeated 3 times. The high static pressure extraction was performed again at a homogenization pressure of 1000 bar and the number of times was homogenized 4 times. After the extraction was completed, the selenium-enriched yeast protein liquid was obtained by centrifugation at a speed of 7000 r / min for 10 min, and the preparation rate was 79.78%.

[0076] Initial separation: the selenium-enriched yeast protein solution was fully stirred, phosphoric acid acid reagent was slowly added dropwise, the pH of the system was adjusted to 3.8, and the adjusted selenium-enriched yeast protein solution was placed in a 4°C environment for protein precipitation for 6 hours, and then centrifuged at 8000r / min for 10 minutes to obtain yeast total selenoprotein, with a preparation yield of 78.57%.

[0077] Sperm separation: the yeast total selenoprotein is dissolved, loaded on a dextran gel chromatography column, and selectively separated, the eluent is a pH=10 phosphate buffer, the flow rate is 1.5 mL / min, and the eluate whose elution volume is 0.15 to 0.67 times the gel filling volume of the dextran gel chromatography column is collected; the eluate contains the sperm selenoprotein.

[0078] The ABTS method specified in the national standard GB / T 39100-2020 was used to determine the antioxidant activity of the selenoprotein (selenoprotein-enriched selenium): 0.02 mL of sample solution and 0.18 mL of ABTS solution were taken respectively, mixed evenly, and reacted at room temperature in the dark for 30 minutes. The absorbance value of the sample at 734 nm was tested by an ultraviolet spectrophotometer (the sample solvent was adjusted to zero). The free radical scavenging rate was calculated to be 79.27%. Under the condition of equal concentration, the antioxidant activity of the selenoprotein was 14.52 times that of the total selenium protein of yeast (non-selenoprotein-enriched selenium total protein) and 13.17 times that of selenium-enriched yeast ( Figure 2 ); The amino acid determination analysis according to GB 5009.124 method determined that the sperm selenoprotein consists of selenomethionine, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, lysine, histidine and selenocysteine ​​( Figure 3 ), the molar proportions are 5.21%, 10.10%, 8.49%, 28.27%, 5.39%, 8.70%, 8.94%, 3.76%, 5.59%, 7.93% and 7.62%, and the molecular weight of the selenoprotein is 36.476KD ( Figure 4 ); The selenium content of the refined selenoprotein was determined to be 4730.47 mg / kg by inductively coupled plasma mass spectrometry.

[0079] Example 4

[0080] Premixing: Weigh 10 g of selenium-enriched yeast, add water according to the ratio of selenium-enriched yeast mass to water volume of 1 g: 10 mL and stir thoroughly. After stirring thoroughly, add potassium hydroxide alkaline reagent dropwise to adjust the system pH to 10 to obtain a selenium-enriched yeast suspension.

[0081] Extraction: The selenium-enriched yeast suspension was subjected to high-pressure homogenization extraction, with a homogenization pressure of 1200 bar and a homogenization time of 3 times. After the extraction, the suspension was centrifuged at a speed of 7000 r / min for 10 min to obtain a selenium-enriched yeast protein solution with a preparation yield of 65.53%.

[0082] Initial separation: the selenium-enriched yeast protein liquid was fully stirred, and malic acid was slowly added to adjust the pH of the system to 4.2. The adjusted selenium-enriched yeast protein liquid was placed in a 4°C environment for protein precipitation for 5 hours, and then centrifuged at 12000 r / min for 10 minutes to obtain total yeast selenoprotein with a preparation yield of 68.43%.

[0083] Sperm separation: the yeast total selenoprotein is dissolved, loaded on a dextran gel chromatography column, and selectively separated, the eluent is a pH=10 phosphate buffer, the flow rate is 1 mL / min, and the eluent whose elution volume is 0.15 to 0.67 times the gel filling volume of the dextran gel chromatography column is collected; the eluent contains the selenoprotein of sperm.

[0084] The ABTS method specified in the national standard GB / T 39100-2020 was used to determine the antioxidant activity of the selenoprotein: 0.02 mL of sample solution and 0.18 mL of ABTS solution were taken respectively, mixed evenly, and reacted at room temperature in the dark for 30 minutes. The absorbance value of the sample at 734 nm was tested by an ultraviolet spectrophotometer (the sample solvent was adjusted to zero), and the free radical scavenging rate was calculated to be 67.48%. Under the same concentration conditions, the antioxidant activity of the selenoprotein was 12.36 times that of the total selenoprotein in yeast and 11.21 times that of selenium-enriched yeast; according to GB The amino acid determination analysis of method 5009.124 determined that the selenoprotein was composed of selenomethionine, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, lysine, histidine and selenocysteine, with each molar proportion being 7.21%, 15.34%, 6.73%, 26.13%, 2.37%, 6.41%, 14.47%, 4.37%, 5.19%, 5.18% and 6.60%. The molecular weight of the selenoprotein was determined to be 39.717KD by gel permeation chromatography; the selenium content of the selenoprotein was determined to be 4120.74mg / kg by inductively coupled plasma mass spectrometry.

[0085] Example 5

[0086] Premixing: Weigh 10g of selenium-enriched yeast, add water according to the ratio of selenium-enriched yeast mass to water volume of 1g:20mL and stir thoroughly. After stirring thoroughly, add potassium hydroxide alkaline reagent dropwise to adjust the system pH to 12 to obtain selenium-enriched yeast suspension.

[0087] Extraction: The selenium-enriched yeast suspension was subjected to high static pressure extraction, the extraction pressure was 150 MPa, the extraction time was 5 min, and the number of repetitions was 3 times. After the extraction, the selenium-enriched yeast protein liquid was obtained by centrifugation at a speed of 6000 r / min for 15 min, and the preparation yield was 62.41%.

[0088] Initial separation: the selenium-enriched yeast protein liquid was fully stirred, and citric acid was slowly added to adjust the system pH to 4.2. The adjusted selenium-enriched yeast protein liquid was placed in a 4°C environment for protein precipitation for 6 hours, and then centrifuged at 12000r / min for 10 minutes to obtain yeast total selenoprotein, with a preparation yield of 68.56%.

[0089] Sperm separation: the yeast total selenoprotein is dissolved, loaded on a dextran gel chromatography column, and selectively separated, the eluent is a pH=10 phosphate buffer, the flow rate is 2 mL / min, and the eluent whose elution volume is 0.15 to 0.67 times the gel filling volume of the dextran gel chromatography column is collected; the eluent contains the selenoprotein.

[0090] The ABTS method specified in the national standard GB / T 39100-2020 was used to determine the antioxidant activity of the selenoprotein: 0.02 mL of sample solution and 0.18 mL of ABTS solution were taken respectively, mixed evenly, and reacted at room temperature in the dark for 30 minutes. The absorbance value of the sample at 734 nm was tested by an ultraviolet spectrophotometer (the sample solvent was adjusted to zero), and the free radical scavenging rate was calculated to be 65.23%. Under the same concentration conditions, the antioxidant activity of the selenoprotein was 11.95 times that of the total selenoprotein in yeast and 10.84 times that of selenium-enriched yeast; according to GB The amino acid determination analysis of method 5009.124 determined that the selenoprotein for selenoprotein consists of selenomethionine, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, lysine, histidine and selenocysteine, with each molar proportion being 3.53%, 14.17%, 7.52%, 27.66%, 5.27%, 8.72%, 9.43%, 4.63%, 4.49%, 6.96% and 7.62%. The molecular weight of the selenoprotein for selenoprotein was 40.717KD as determined by gel permeation chromatography; the selenium content of the selenoprotein for selenoprotein was 3987.59mg / kg as determined by inductively coupled plasma mass spectrometry.

[0091] Example 6

[0092] Premixing: Weigh 10g of selenium-enriched yeast, add water according to the ratio of selenium-enriched yeast mass to water volume of 1g:10mL and stir thoroughly. After stirring thoroughly, add sodium carbonate alkaline reagent dropwise to adjust the system pH to 9.5 to obtain selenium-enriched yeast suspension.

[0093] Extraction: The selenium-enriched yeast suspension was subjected to high-pressure homogenization extraction at a homogenization pressure of 1500 bar and a homogenization time of 2 times. After the extraction, the suspension was centrifuged at a speed of 10000 r / min for 10 min to obtain a selenium-enriched yeast protein solution with a preparation yield of 64.47%.

[0094] Initial separation: the selenium-enriched yeast protein solution was fully stirred, phosphoric acid acid reagent was slowly added dropwise, the pH of the system was adjusted to 3.8, and the adjusted selenium-enriched yeast protein solution was placed in a 4°C environment for protein precipitation for 6 hours, and then centrifuged at 12000r / min for 10 minutes to obtain yeast total selenoprotein, with a preparation yield of 71.63%.

[0095] Sperm separation: the yeast total selenoprotein is dissolved, loaded on a dextran gel chromatography column, and selectively separated, the eluent is a pH=10 phosphate buffer, the flow rate is 1.8 mL / min, and the eluate whose elution volume is 0.15 to 0.67 times the gel filling volume of the dextran gel chromatography column is collected; the eluate contains the sperm selenoprotein.

[0096] The ABTS method specified in the national standard GB / T 39100-2020 was used to determine the antioxidant activity of the selenoprotein: 0.02 mL of sample solution and 0.18 mL of ABTS solution were taken respectively, mixed evenly, and reacted at room temperature in the dark for 30 minutes. The absorbance value of the sample at 734 nm was tested by an ultraviolet spectrophotometer (the sample solvent was adjusted to zero), and the free radical scavenging rate was calculated to be 70.41%. Under the same concentration conditions, the antioxidant activity of the selenoprotein was 12.90 times that of the total selenoprotein in yeast and 11.70 times that of selenium-enriched yeast; according to GB The amino acid determination analysis of method 5009.124 determined that the selenoprotein for selenoprotein consists of selenomethionine, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, lysine, histidine and selenocysteine, with each molar proportion being 3.97%, 13.37%, 7.63%, 27.87%, 4.73%, 6.98%, 9.94%, 4.39%, 4.63%, 6.87% and 9.62%. The molecular weight of the selenoprotein for selenoprotein was 38.113KD as determined by gel permeation chromatography; the selenium content of the selenoprotein for selenoprotein was 4237.13mg / kg as determined by inductively coupled plasma mass spectrometry.

[0097] Comparative Example 1

[0098] Weigh 10g of selenium-enriched yeast, add water according to the ratio of selenium-enriched yeast mass to water volume of 1g:10mL and stir thoroughly. After stirring thoroughly, add sodium carbonate alkaline reagent dropwise to adjust the system pH to 9.5 to obtain a selenium-enriched yeast suspension.

[0099] The selenium-enriched yeast suspension was subjected to high-pressure homogenization extraction at a homogenization pressure of 1000 bar for 15 minutes. After the extraction, the suspension was centrifuged at a speed of 10000 r / min for 10 minutes to obtain a selenium-enriched yeast protein solution with a preparation yield of 74.47%.

[0100] The selenium-enriched yeast total protein solution was fully stirred, and a phosphoric acid acid reagent was slowly added dropwise to adjust the system pH to 4.0. The adjusted selenium-enriched yeast protein solution was placed in a 4° C. environment for protein precipitation for 3 hours, and then centrifuged at a speed of 12000 r / min for 10 minutes to obtain selenium-enriched yeast protein with a preparation yield of 89.63%.

[0101] The ABTS method specified in the national standard GB / T 39100-2020 was used to determine the antioxidant activity of the selenium-enriched yeast protein: 0.02 mL of sample solution and 0.18 mL of ABTS solution were taken respectively, mixed evenly, and reacted at room temperature in the dark for 30 minutes. The free radical scavenging rate was calculated to be 5.46% by the absorbance value of the sample at 734 nm (sample solvent was adjusted to zero) by an ultraviolet spectrophotometer; The amino acid determination and analysis of the selenium-enriched yeast protein by the 5009.124 method showed that the selenium-enriched yeast protein consisted of selenomethionine, proline, valine, aspartic acid, phenylalanine, selenocysteine, serine, lysine, histidine, threonine, serine, glutamic acid, glycine and alanine, with the molar proportions of 0.75%, 21.72%, 1.48%, 23.66%, 2.04%, 1.48%, 4.35%, 21.22%, 2.78%, 5.73%, 3.48%, 6.37%, 3.21% and 1.73% respectively. The molecular weight of the selenium-enriched yeast protein was determined to be 108.664KD by gel permeation chromatography; the selenium content of the selenium-enriched yeast protein was determined to be 709.29mg / kg by inductively coupled plasma mass spectrometry.

[0102] In summary, the selenoprotein prepared by the present invention is a selenoprotein substance with more outstanding yeast-derived high biological activity than selenium-enriched yeast, with an ABTS free radical scavenging rate of 55.03% to 79.27%, a selenium content of 3498.09 to 4730.47 mg / kg, and mild reaction conditions for preparation with high selectivity.

[0103] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A selenoprotein with high antioxidant activity, characterized in that The molecular weight of the selenoprotein with high antioxidant activity is 36.476-42.117 KD, and the selenium content is 3498.09-4730.47 mg / kg.

2. The selenoprotein with high antioxidant activity according to claim 1, characterized in that The high antioxidant activity selenoprotein is composed of the following amino acids in molar percentage: 3.47% to 7.21% selenomethionine, 10.10% to 15.34% aspartic acid, 6.73% to 8.49% threonine, 26.13% to 28.27% serine, 2.37% to 5.39% glutamic acid, 6.41% to 8.72% glycine, 7.33% to 15.94% alanine, 2.85% to 4.76% valine, 4.31% to 5.59% lysine, 5.18% to 7.93% histidine and 6.54% to 9.62% selenocysteine.

3. The selenoprotein with high antioxidant activity according to claim 2, characterized in that The selenoprotein with the highest antioxidant activity is composed of the following amino acids in molar percentage: 5.21% selenomethionine, 10.10% aspartic acid, 8.49% threonine, 28.27% serine, 5.39% glutamic acid, 8.70% glycine, 8.94% alanine, 3.76% valine, 5.59% lysine, 7.93% histidine and 7.62% selenocysteine.

4. The method for preparing the selenoprotein with high antioxidant activity according to any one of claims 1 to 3, characterized in that: The following steps are involved: Mixing selenium-enriched yeast and water to obtain a selenium-enriched yeast suspension; Extracting selenoproteins from the selenium-enriched yeast suspension to obtain a selenoprotein extract; the extraction method includes: high-pressure homogenization extraction and / or high-static pressure extraction; Adjusting the pH value of the selenoprotein extract to acidic to perform protein precipitation to obtain a precipitate; The precipitate is separated by a dextran gel chromatography column, eluted with a phosphate buffer solution of pH=10, and an eluate having an elution volume multiple of 0.15 to 0.67 times the gel filling volume of the dextran gel chromatography column is collected, wherein the eluate contains the selenoprotein with high antioxidant activity.

5. The preparation method according to claim 4, characterized in that: During the high-pressure homogenization extraction, the homogenization pressure is 800-1500 bar, and the homogenization times are 1-8 times; During the high static pressure extraction, the extraction pressure is 50-200 MPa, the extraction times are 1-3 times, and the time of each extraction is 3-5 minutes.

6. The preparation method according to claim 4 or 5, characterized in that: The extraction method is a combination of high static pressure extraction and high pressure homogenization extraction.

7. The preparation method according to claim 4, characterized in that: The flow rate of the elution is 0.5 to 3 mL / min.

8. The preparation method according to claim 4, characterized in that: The pH value of the selenoprotein extract is adjusted to 3-4.5 for protein precipitation; the temperature of the protein precipitation is 1-4°C.

9. The preparation method according to claim 4, characterized in that: Before extracting the selenium-enriched yeast suspension, the pH value of the selenium-enriched yeast suspension is adjusted to 9.5-12.

10. Use of the selenoprotein with high antioxidant activity according to any one of claims 1 to 3 or the yeast selenoprotein prepared by the preparation method according to any one of claims 4 to 9 in selenium supplementation or preparation of selenium supplementation agent.

Images