Method for extracting selenoprotein from selenium-rich chlorella pyrenoidosa

Abstract

This invention relates to the field of microbial active ingredient extraction technology, and discloses a method for extracting selenoproteins from selenium-rich Chlorella. The method includes selenium-tolerant algal strain domestication, selenium-enriched culture, collection and processing, compound cell wall disruption, alkali extraction, acid precipitation, purification, and drying. The method utilizes gradient selenium stress combined with UV mutagenesis to domesticate the algal strain, enhancing its selenium tolerance and enrichment capacity, thereby increasing the conversion of organic selenium from the source. A modified culture medium and phased control of culture conditions result in high selenium conversion efficiency and a short cycle. Enzymatic hydrolysis-ultrasonic compound cell wall disruption is employed for efficient disruption while preserving selenoprotein activity, improving extraction yield. Multiple steps of deep impurity removal, including alkali dissolution, acid precipitation, decolorization, salting out, and dialysis, effectively remove various impurities, resulting in a high-purity finished product. Low-temperature, mild acid-base, and vacuum freeze-drying treatments are used throughout the process to maximize protein activity retention. The entire process is streamlined, easy to operate, and produces stable product quality, suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of microbial active ingredient extraction technology, specifically a method for extracting selenoproteins from selenium-rich Chlorella nucleatum. Background Technology

[0002] Selenium is one of the essential trace elements for the human body. It has a variety of physiological functions such as anti-oxidation, enhancing the body's immunity, and antagonizing heavy metal toxicity. Chlorella proteoglycans has a fast reproduction rate, mild culture conditions, and large biomass. It also has a strong ability to enrich and biotransform substances, making it an ideal carrier for preparing natural organic selenium protein.

[0003] Currently, there are still many shortcomings in the industry regarding the preparation of selenoproteins using Chlorella: First, Chlorella vulgaris has low tolerance to selenium, and the algae are easily inactivated and their growth is inhibited under high selenium concentrations, resulting in insufficient selenium accumulation and low organic selenium content in the final product; Second, the cell wall structure of Chlorella vulgaris is dense, and the single cell wall disruption method has a low disruption rate, resulting in insufficient release of intracellular selenoproteins and poor extraction yield; Third, conventional extraction processes only use simple leaching and precipitation treatments, resulting in a large amount of phycochromes, polysaccharides, inorganic selenium, and other proteins mixed in the product, leading to low purity of selenoproteins and limiting subsequent applications.

[0004] In view of this, and in response to the above problems, an in-depth study was conducted, and a method for extracting selenoproteins from selenium-rich Chlorella nucleatum was proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting selenoproteins from selenium-rich Chlorella nucleatum, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting selenoproteins from selenium-rich Chlorella nucleatum, comprising the following steps:

[0007] Step A, selenium-tolerant algal strain domestication: Select the original Chlorella proteoglycan strain, and use gradient selenium stress domestication combined with ultraviolet mutagenesis screening to obtain domesticated algal strains with high selenium tolerance and high accumulation capacity.

[0008] Step B, Selenium-enriched culture: The domesticated algal strains are inoculated into a modified heterotrophic fermentation medium, and sodium selenite is added for selenium-enriched culture to obtain a selenium-enriched Chlorella protein-nucleated fermentation broth.

[0009] Step C, Collection and Processing: Centrifuge, wash, and dry the selenium-enriched protein-nucleated Chlorella fermentation broth at low temperature, then pulverize and sieve to obtain selenium-enriched Chlorella dry powder, which is then stored in a sealed container at low temperature for later use.

[0010] Step D, Composite Cell Wall Disruption Treatment: Selenium-enriched Chlorella dry powder is mixed with pre-cooled buffer to prepare algal suspension. Enzymatic hydrolysis-ultrasound composite cell wall disruption process is used to break down the cell walls of algae in steps and release intracellular selenoproteins.

[0011] Step E, Alkali Extraction: Adjust the algal suspension after cell wall disruption to an alkaline environment, stir and extract at a constant temperature to fully dissolve the selenoprotein, and centrifuge to obtain the crude selenoprotein supernatant.

[0012] Step F, acid precipitation of protein: Adjust the pH of the crude extract supernatant to acidic at low temperature, let it stand at a constant temperature to precipitate, and collect the crude precipitate of selenoprotein by centrifugation;

[0013] Step G, Purification and Impurity Removal: A stepwise purification process is used to remove pigments, polysaccharides, inorganic selenium and small molecule impurities to obtain a purified selenium protein solution;

[0014] Step H, Drying and Powdering: The refined selenium protein liquid is freeze-dried under low temperature and vacuum, then pulverized and sieved to obtain a high-purity selenium-enriched Chlorella selenium protein product.

[0015] The above technical solution facilitates the efficient preparation of high-purity, high-activity Chlorella selenium protein with good extraction results.

[0016] As a preferred technical solution of the present invention, the gradient selenium stress acclimatization in step A specifically involves: sequentially transferring the original Chlorella proteoglycans strain to basal culture media with sodium selenite concentration gradients of 100 mg / L, 300 mg / L, 500 mg / L, 800 mg / L, and 1500 mg / L, and acclimatizing in the dark for 5-7 days at each gradient to gradually improve the selenium tolerance of the algal strain; the ultraviolet mutagenesis screening specifically involves: spreading the acclimatized algal solution onto a plate, mutagenesis treatment at 30-40 cm under an ultraviolet lamp for 30-60 seconds, followed by dark incubation for 12 hours to repair, and screening for single colonies with excellent growth, which are the highly selenium-tolerant acclimatized algal strains.

[0017] The above technical solution facilitates the gradual domestication of algal strains under gradient selenium stress, avoiding the inactivation of algal strains due to a sudden increase in selenium concentration. Combined with UV mutagenesis with precise parameters, it enables targeted screening of algal strains with excellent selenium tolerance and enrichment capabilities, resulting in a high screening success rate and stable algal strain traits.

[0018] As a preferred technical solution of the present invention, in step B, the domesticated algal strain is inoculated into a modified heterotrophic fermentation medium with an inoculation volume fraction of 8% to 12%, and cultured at a temperature of 25 to 30°C and a pH of 5.5 to 6.0 for 2 to 3 days in the dark; sodium selenite is added at a concentration of 2 to 4 mg / L, the pH is adjusted to 6.0 to 7.0, the temperature is maintained at 25 to 30°C, and the culture is continued for another 2 to 3 days to complete the biotransformation of inorganic selenium into organic selenoprotein.

[0019] The modified heterotrophic fermentation medium formula is as follows: glucose 20 g / L, peptone 6 g / L, sodium nitrate 2 g / L, dipotassium hydrogen phosphate 0.08 g / L, magnesium sulfate heptahydrate 0.05 g / L, calcium chloride 0.01 g / L, sodium carbonate 0.04 g / L, citric acid 0.002 g / L, ferric citrate 0.003 g / L, trace element solution 1 g / L, sodium chloride 18 g / L, and the balance being distilled water.

[0020] The above technical solution facilitates staged temperature and pH control culture, which meets the needs of both algal growth and selenium conversion. The culture medium has a scientific ratio of carbon source, nitrogen source and trace elements, which can provide sufficient nutrients for the growth of Chlorella and the bioconversion of selenium, with a short conversion cycle and high conversion efficiency.

[0021] As a preferred technical solution of the present invention, in step C, the centrifugation speed is 5000-6000 rpm and the centrifugation time is 8-12 min, and the product is washed 2-3 times with sterile distilled water; the drying temperature is 40-45℃ and the drying time is 8-12 h, and the product is pulverized and passed through an 80-100 mesh sieve.

[0022] The above technical solution allows for complete retention of algae and reduced bacterial damage, aseptic washing to remove fermentation residue, and low-temperature constant-temperature drying to balance drying efficiency and protein activity. After sieving, the material has a uniform particle size, making subsequent material mixing and cell wall breaking processes more uniform.

[0023] As a preferred technical solution of the present invention, step D specifically includes:

[0024] Step D1, Mixing: Mix the selenium-enriched Chlorella powder with a phosphate buffer solution of pH 7.0-7.4 at a ratio of 1:12 to 1:18 until homogeneous;

[0025] Step D2, Enzymatic hydrolysis: Add a compound cell wall-breaking enzyme and hydrolyze at a constant temperature of 40-50℃ for 2-3 hours. The compound cell wall-breaking enzyme is a mixture of cellulase, pectinase, and snail enzyme. The total amount of the compound cell wall-breaking enzyme added is 1.5%-3.0% of the mass of the selenium-enriched Chlorella dry powder.

[0026] Step D3, Ultrasound: Turn on ultrasound to facilitate cell wall disruption. The ultrasound power is 200-300W and the ultrasound frequency is 20-25kHz. The ultrasound works for 3 seconds, with a 5-second interval, and is cycled for 15-25 minutes to complete the composite cell wall disruption.

[0027] The above technical solution facilitates the protection of enzyme activity and protein structure by neutral buffer system, and the combination of multiple enzymes can specifically decompose different components of cell wall. Intermittent ultrasound avoids local high temperature damage to selenoproteins. The synergistic effect of the two results in a cell wall disruption rate that is much higher than that of a single process, and the release of selenoproteins is more thorough.

[0028] As a preferred embodiment of the present invention, in step E, the pH of the algal suspension is adjusted to 8.5-9.5 using 0.1 mol / L sodium hydroxide solution, and the suspension is stirred at room temperature for 1.5-2.5 hours at a stirring speed of 150-200 rpm; the centrifugation speed is 6000 rpm and the time is 10-15 minutes.

[0029] The above technical solution facilitates efficient dissolution of selenoproteins in a mild alkaline environment without causing protein denaturation. The constant stirring speed ensures uniform mixing of the extraction system, and the centrifugation parameters are adapted to the system characteristics, resulting in thorough solid-liquid separation and reduced selenoprotein loss.

[0030] As a preferred technical solution of the present invention, in step F, the crude extract supernatant is cooled to 4-10°C, the pH is slowly adjusted to 4.0-5.0 with glacial acetic acid, and the mixture is allowed to stand at low temperature for 2-4 hours to precipitate; the mixture is then centrifuged at 6000 rpm for 10 minutes, the supernatant is discarded, and the grayish-white selenoprotein crude precipitate is collected.

[0031] The above technical solution facilitates the inhibition of protein degradation in a low-temperature environment, and the slow adjustment of pH with glacial acetic acid can avoid damage to the protein due to excessively high local acid and alkali concentrations. The selenoprotein precipitate is dense and has a high collection rate.

[0032] As a preferred embodiment of the present invention, the stepwise purification process in step G specifically includes:

[0033] Step G1, Decolorization: Add 85% to 95% anhydrous ethanol (by volume) to the crude selenoprotein precipitate at a ratio of 1:8 to 1:12. Stir and decolorize at room temperature for 40 to 60 minutes, centrifuge at 6000 rpm for 10 minutes, discard the decolorization waste liquid, and repeat the decolorization process twice to remove phycobilichrome and lipid-soluble impurities.

[0034] Step G2, Resolution and Removal: Resolute the decolorized protein precipitate with phosphate buffer, add ammonium sulfate for fractional salting out, control the saturation at 30% to 50%, let stand for 1 hour, and centrifuge to remove impurities such as proteins and polysaccharides.

[0035] Step G3, dialysis desalting: The salted-out protein solution is placed into a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed at 4°C for 12-24 hours. The dialysis solution is changed every 4 hours to completely remove inorganic selenium, salt and small molecule impurities, and obtain a purified selenoprotein solution.

[0036] The above technical solution facilitates targeted removal of pigments and lipid impurities by ethanol decolorization, separation of impurities such as proteins and polysaccharides by graded salting out, and precise removal of inorganic selenium, salts and small molecules by dialysis. The combination of multiple processes achieves deep impurity removal and significantly improves the purity of the finished product.

[0037] As a preferred technical solution of the present invention, in step H, the vacuum degree is 0.08~0.1MPa, the freezing temperature is -40~-35℃, the vacuum freeze-drying is carried out for 24~36h, and the powder is passed through a 100-mesh sieve.

[0038] By adopting the above technical solution, low-temperature vacuum freeze drying can maximize the preservation of selenoprotein bioactivity, avoid nutrient loss and protein inactivation caused by high-temperature drying, and the final product powder is fine, has good solubility, and stable quality.

[0039] Compared with the prior art, the beneficial effects of the present invention are: the method for extracting selenoprotein from Chlorella vulgaris; 1. The algal strains were domesticated by using gradient selenium stress combined with ultraviolet mutagenesis to gradually improve their selenium tolerance and effectively avoid algal inactivation under high selenium conditions. The domesticated algal strains obtained by screening have outstanding selenium enrichment capacity, which improves the conversion of organic selenium and the quality of raw materials from the source. 2. The enzymatic hydrolysis-ultrasound composite cell wall disruption technology, combined with a suitable buffer system and intermittent ultrasound mode, can not only fully decompose the dense cell wall structure of Chlorella and greatly improve the cell wall disruption rate, but also avoid the denaturation and inactivation of selenoproteins caused by local high temperature and strong mechanical action, so as to release intracellular selenoproteins more fully and effectively improve the extraction yield. 3. Through a series of purification processes including alkaline extraction, low-temperature acid precipitation, ethanol decolorization, fractional salting out, and low-temperature dialysis, phycobilichromes, fat-soluble substances, impurities, polysaccharides, inorganic selenium, and various small molecule impurities can be removed in layers, achieving deep impurity removal and significantly improving the purity of the selenoprotein product. At the same time, a mild acid-base environment, low-temperature treatment, and vacuum freeze-drying are used throughout the process to maximize the preservation of the biological activity of the selenoprotein and prevent the loss of activity caused by high temperature and strong acid-base conditions. 4. By improving the heterotrophic fermentation medium, the culture temperature, pH and culture time are controlled in stages, and the nutrient ratio is scientific, which can meet the dual needs of algal growth and inorganic selenium bioconversion, with a short conversion cycle and high conversion efficiency. 5. The overall process is coherent and highly operable, and the final selenium protein powder is fine, has good solubility, and stable quality. The overall production effect is far superior to that of traditional processes, making it suitable for large-scale production applications. Attached Figure Description

[0040] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1

[0043] The technical solution of this invention: A method for extracting selenoproteins from selenium-rich Chlorella vulgaris, comprising the following steps:

[0044] Step A: Domestication of Selenium-Tolerant Algal Strains: Select the original Chlorella proteoglycan strain and transfer it sequentially to basal culture media with sodium selenite concentration gradients of 100 mg / L, 300 mg / L, 500 mg / L, 800 mg / L, and 1500 mg / L. Each gradient is cultured in the dark for 5 days to gradually improve the selenium tolerance of the algal strain. Spread the domesticated algal solution onto plates and induce mutagenesis at 30 cm under UV light for 60 seconds. Then, incubate in the dark for 12 hours to regenerate. Select single colonies with excellent growth to obtain domesticated algal strains with high selenium tolerance and high accumulation capacity.

[0045] Step B, Selenium-enriched culture: The domesticated algal strain was inoculated into a modified heterotrophic fermentation medium with an inoculation volume fraction of 8%. The culture temperature was 25℃, pH 5.5, and the culture was carried out in the dark for 2 days. 2 mg / L sodium selenite was added to adjust the pH to 6.0 and the temperature to 25℃. The culture was continued for another 2 days to complete the biotransformation of inorganic selenium into organic selenoprotein, obtaining a selenium-enriched protein Chlorella fermentation broth. The modified heterotrophic fermentation medium formula was as follows: 20 g / L glucose, 6 g / L peptone, 2 g / L sodium nitrate, 0.08 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate heptahydrate, 0.01 g / L calcium chloride, 0.04 g / L sodium carbonate, 0.002 g / L citric acid, 0.003 g / L ferric citrate, 1 g / L trace element solution, 18 g / L sodium chloride, and the remainder was distilled water.

[0046] Step C, Collection and Processing: Centrifuge the fermentation broth of selenium-enriched protein-nucleated Chlorella at 5000 rpm for 12 min, wash twice with sterile distilled water; dry at 40℃ for 12 h, pulverize and pass through an 80-mesh sieve to obtain selenium-enriched Chlorella dry powder, and store in a sealed container at low temperature for later use.

[0047] Step D: Compound cell wall disruption treatment:

[0048] Step D1, Mixing: Mix the selenium-enriched Chlorella powder with pH 7.0 phosphate buffer at a ratio of 1:12 until homogeneous;

[0049] Step D2, Enzymatic hydrolysis: Add a compound cell wall-breaking enzyme and hydrolyze at a constant temperature of 40℃ for 3 hours. The compound cell wall-breaking enzyme is a mixture of cellulase, pectinase, and snail enzyme. The total amount of the compound cell wall-breaking enzyme added is 1.5% of the mass of the selenium-enriched Chlorella powder.

[0050] Step D3, Ultrasound: Turn on ultrasound-assisted cell disruption, with an ultrasound power of 200W and an ultrasound frequency of 25kHz. The ultrasound will run for 3 seconds, with a 5-second interval, and the process will be repeated for 15 minutes to complete the composite cell disruption.

[0051] Step E, Alkali extraction: Adjust the pH of the algal suspension to 8.5 using 0.1 mol / L sodium hydroxide solution, stir at room temperature for 1.5 h at a stirring speed of 200 rpm to fully dissolve the selenoprotein, and centrifuge at 6000 rpm for 10 min to obtain the crude selenoprotein supernatant.

[0052] Step F, acid precipitation of protein: Cool the crude extract supernatant to 4°C, slowly adjust the pH to 4.0 with glacial acetic acid, and let it stand at low temperature for 2 hours to precipitate; centrifuge at 6000 rpm for 10 minutes, discard the supernatant, and collect the grayish-white crude selenoprotein precipitate.

[0053] Step G: Purification and impurity removal: Step G1, Decolorization: Add 85% anhydrous ethanol (by volume) to the crude selenoprotein precipitate at a ratio of 1:8. Stir and decolorize at room temperature for 40 min, centrifuge at 6000 rpm for 10 min, discard the decolorization waste liquid, and repeat the decolorization process twice to remove phycobilichrome and lipid-soluble impurities. Step G2, Resolution and Removal: Resolute the decolorized protein precipitate with phosphate buffer, add ammonium sulfate for fractional salting out, control the saturation at 30%, let stand for 1 hour, and centrifuge to remove impurities such as proteins and polysaccharides. Step G3, Dialysis Desalting: The protein solution after salting out is placed into a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed at 4°C for 12 hours. The dialysis solution is changed every 4 hours to completely remove inorganic selenium, salt and small molecule impurities, and obtain a refined selenium protein solution. Step H, Drying and Powdering: The refined selenium protein solution is vacuum freeze-dried for 36 hours under a vacuum of 0.08 MPa and a freezing temperature of -40℃. After pulverizing, it is passed through a 100-mesh sieve to obtain a high-purity selenium-enriched Chlorella selenoprotein product.

[0054] Example 2

[0055] The technical solution of this invention: A method for extracting selenoproteins from selenium-rich Chlorella vulgaris, comprising the following steps: Step A: Domestication of Selenium-Tolerant Algal Strains: Select the original Chlorella proteoglycan strain and transfer it sequentially to basal culture media with sodium selenite concentration gradients of 100 mg / L, 300 mg / L, 500 mg / L, 800 mg / L, and 1500 mg / L. Each gradient is cultured in the dark for 6 days to gradually improve the selenium tolerance of the algal strain. Spread the domesticated algal solution on a plate and inducing mutagenesis at 35 cm under a UV lamp for 45 s. Then, incubate in the dark for 12 h to repair the damage. Select single colonies with excellent growth to obtain domesticated algal strains with high selenium tolerance and high accumulation capacity. Step B, Selenium-enriched culture: The domesticated algal strain was inoculated into a modified heterotrophic fermentation medium with an inoculation volume fraction of 10%. The culture temperature was 28℃, pH 5.8, and the culture was carried out in the dark for 2 days. 3 mg / L sodium selenite was added to adjust the pH to 6.5 and the temperature to 27℃. The culture was continued for 3 days to complete the biotransformation of inorganic selenium into organic selenoprotein, obtaining a selenium-enriched protein Chlorella fermentation broth. The modified heterotrophic fermentation medium formula was as follows: 20 g / L glucose, 6 g / L peptone, 2 g / L sodium nitrate, 0.08 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate heptahydrate, 0.01 g / L calcium chloride, 0.04 g / L sodium carbonate, 0.002 g / L citric acid, 0.003 g / L ferric citrate, 1 g / L trace element solution, 18 g / L sodium chloride, and the remainder was distilled water.

[0056] Step C, Collection and Processing: Centrifuge the fermentation broth of selenium-enriched protein-nucleated Chlorella at 5500 rpm for 10 min, wash it three times with sterile distilled water, dry it at 43℃ for 10 h, pulverize it and pass it through a 90-mesh sieve to obtain selenium-enriched Chlorella dry powder, and store it in a sealed container at low temperature for later use. Step D: Compound cell wall disruption treatment: Step D1, Mixing: Mix the selenium-enriched Chlorella powder with a pH 7.2 phosphate buffer solution at a ratio of 1:15 until homogeneous; Step D2, Enzymatic hydrolysis: Add a compound cell wall-breaking enzyme and hydrolyze at a constant temperature of 45℃ for 2.5 hours. The compound cell wall-breaking enzyme is a mixture of cellulase, pectinase, and snail enzyme. The total amount of the compound cell wall-breaking enzyme added is 2% of the mass of the selenium-enriched Chlorella powder. Step D3, Ultrasound: Turn on ultrasound-assisted cell disruption, with an ultrasound power of 260W and an ultrasound frequency of 22kHz. The ultrasound will run for 3 seconds, with a 5-second interval, and the process will be repeated for 20 minutes to complete the composite cell disruption.

[0057] Step E, Alkali extraction: Adjust the pH of the algal suspension to 9.1 using 0.1 mol / L sodium hydroxide solution, stir at room temperature for 2 hours at a stirring speed of 180 rpm to fully dissolve the selenoprotein, and centrifuge at 6000 rpm for 12 minutes to obtain the crude selenoprotein supernatant. Step F, acid precipitation of protein: Cool the crude extract supernatant to 7°C, slowly adjust the pH to 4.5 with glacial acetic acid, and let it stand at low temperature for 3 hours to precipitate; centrifuge at 6000 rpm for 10 minutes, discard the supernatant, and collect the grayish-white crude selenoprotein precipitate. Step G: Purification and impurity removal: Step G1, Decolorization treatment: Add 90% anhydrous ethanol (by volume) to the crude selenoprotein precipitate at a material-to-liquid ratio of 1:10. Stir at room temperature for 50 min to decolorize, centrifuge at 6000 rpm for 10 min, discard the decolorization waste liquid, and repeat the decolorization process twice to remove phycobilichrome and lipid-soluble impurities. Step G2, Resolution and Removal: Resolute the decolorized protein precipitate with phosphate buffer, add ammonium sulfate for fractional salting out, control the saturation at 40%, let stand for 1 hour, and centrifuge to remove impurities such as proteins and polysaccharides. Step G3, Dialysis Desalting: The protein solution after salting out is placed into a dialysis bag with a molecular weight cutoff of 11000 Da and dialyzed at 4°C for 18 hours. The dialysis solution is changed every 4 hours to completely remove inorganic selenium, salt and small molecule impurities, and obtain a refined selenium protein solution. Step H, Drying and Powdering: The refined selenium protein solution is vacuum freeze-dried for 30 hours under a vacuum of 0.09 MPa and a freezing temperature of -38℃. After pulverizing, it is passed through a 100-mesh sieve to obtain a high-purity selenium-enriched Chlorella selenium protein product.

[0058] Example 3

[0059] The technical solution of this invention: A method for extracting selenoproteins from selenium-rich Chlorella vulgaris, comprising the following steps:

[0060] Step A: Domestication of Selenium-Tolerant Algal Strains: Select the original Chlorella proteoglycans strain and transfer it sequentially to basal culture media with sodium selenite concentration gradients of 100 mg / L, 300 mg / L, 500 mg / L, 800 mg / L, and 1500 mg / L. Each gradient is cultured in the dark for 7 days to gradually improve the selenium tolerance of the algal strains. Spread the domesticated algal solution on plates and induce mutagenesis at 40 cm under a UV lamp for 30 seconds. Then, incubate in the dark for 12 hours to repair the damage. Select single colonies with excellent growth to obtain domesticated algal strains with high selenium tolerance and high accumulation capacity.

[0061] Step B, Selenium-enriched culture: The domesticated algal strain was inoculated into a modified heterotrophic fermentation medium with an inoculation volume fraction of 12%. The culture temperature was 30℃, pH 6.0, and the culture was carried out in the dark for 3 days. 4 mg / L sodium selenite was added to adjust the pH to 7.0 and the temperature to 30℃. The culture was continued for another 3 days to complete the biotransformation of inorganic selenium into organic selenoprotein, obtaining a selenium-enriched protein Chlorella fermentation broth. The modified heterotrophic fermentation medium formula was as follows: 20 g / L glucose, 6 g / L peptone, 2 g / L sodium nitrate, 0.08 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate heptahydrate, 0.01 g / L calcium chloride, 0.04 g / L sodium carbonate, 0.002 g / L citric acid, 0.003 g / L ferric citrate, 1 g / L trace element solution, 18 g / L sodium chloride, and the remainder was distilled water.

[0062] Step C, Collection and Processing: Centrifuge the fermentation broth of selenium-enriched protein-nucleated Chlorella at 6000 rpm for 8 min, wash it 3 times with sterile distilled water, dry it at 45℃ for 8 h, pulverize it and pass it through a 100-mesh sieve to obtain selenium-enriched Chlorella dry powder, and store it in a sealed container at low temperature for later use.

[0063] Step D: Compound cell wall disruption treatment:

[0064] Step D1, Mixing: Mix the selenium-enriched Chlorella powder with a pH 7.4 phosphate buffer solution at a ratio of 1:18 until homogeneous;

[0065] Step D2, Enzymatic hydrolysis: Add a compound cell wall-breaking enzyme and hydrolyze at 50℃ for 2 hours. The compound cell wall-breaking enzyme is a mixture of cellulase, pectinase, and snail enzyme. The total amount of the compound cell wall-breaking enzyme added is 3.0% of the mass of the selenium-enriched Chlorella powder.

[0066] Step D3, Ultrasound: Turn on ultrasound-assisted cell disruption, with an ultrasound power of 300W and an ultrasound frequency of 20kHz. The ultrasound will run for 3 seconds, with a 5-second interval, and the process will be repeated for 25 minutes to complete the composite cell disruption.

[0067] Step E, alkaline extraction: The pH of the algal suspension was adjusted to 9.5 using 0.1 mol / L sodium hydroxide solution, and stirred at room temperature for 2.5 h at a stirring speed of 150 rpm to fully dissolve the selenoprotein. The mixture was then centrifuged at 6000 rpm for 15 min to obtain the crude selenoprotein supernatant.

[0068] Step F, acid precipitation of protein: Cool the crude extract supernatant to 10°C, slowly adjust the pH to 5.0 with glacial acetic acid, and let it stand at low temperature for 4 hours to precipitate; centrifuge at 6000 rpm for 10 minutes, discard the supernatant, and collect the grayish-white crude selenoprotein precipitate.

[0069] Step G: Purification and impurity removal:

[0070] Step G1, Decolorization: Add 95% anhydrous ethanol (by volume) to the crude selenoprotein precipitate at a material-to-liquid ratio of 1:12. Stir and decolorize at room temperature for 60 min, centrifuge at 6000 rpm for 10 min, discard the decolorization waste liquid, and repeat the decolorization process twice to remove phycobilichrome and lipid-soluble impurities.

[0071] Step G2, Resolution and Removal: Resolute the decolorized protein precipitate with phosphate buffer, add ammonium sulfate for fractional salting out, control the saturation at 50%, let stand for 1 hour, and centrifuge to remove impurities such as proteins and polysaccharides.

[0072] Step G3, Dialysis Desalting: The salted-out protein solution is placed into a dialysis bag with a molecular weight cutoff of 14000 Da and dialyzed at 4°C for 24 hours. The dialysis solution is changed every 4 hours to completely remove inorganic selenium, salt and small molecule impurities, and obtain a refined selenoprotein solution.

[0073] Step H, Drying and Powdering: The refined selenium protein solution is vacuum freeze-dried for 36 hours under a vacuum of 0.1 MPa and a freezing temperature of -35℃. After pulverizing, it is passed through a 100-mesh sieve to obtain a high-purity selenium-enriched Chlorella selenium protein product.

[0074] Comparative Example 1 (Traditional process: no algae culture domestication + single ultrasonic cell disruption + simple impurity removal)

[0075] The original, undomestic Chlorella proteolytica strain was used, without gradient selenium stress or UV mutagenesis; the selenium-enriched culture was carried out using ordinary basal medium with sodium selenite added at 3 mg / L, and under conventional light for 5 days.

[0076] The collection and processing parameters were the same as in Example 2; only a single ultrasonic cell disruption was used (parameters were the same as the ultrasonic conditions in Example 2, without the enzymatic hydrolysis step).

[0077] The extraction stage only involves alkali dissolution and acid precipitation, omitting the ethanol decolorization, fractional salting, and dialysis desalination processes, and only simple centrifugation to remove impurities; finally, conventional hot air drying (55℃, 20h) is used to pulverize the product, and the remaining operating parameters are consistent with those in Example 2, and the selenoprotein sample is finally prepared.

[0078] Comparative Example 2 (Existing conventional process: algal strain domestication + single enzymatic hydrolysis to break cell walls + simplified purification)

[0079] The algal strain domestication, selenium-enriched culture, and collection and processing parameters are exactly the same as in Example 2;

[0080] The cell wall disruption process only uses compound enzymatic hydrolysis (parameters are the same as the enzymatic hydrolysis conditions in Example 2, without ultrasonic synergy).

[0081] The purification process only involves a single ethanol decolorization and basic salting out, omitting the low-temperature dialysis step; the drying method is vacuum freeze drying (parameters are the same as in Example 2), and the parameters of the remaining steps are consistent with those in Example 2, to prepare selenoprotein samples.

[0082] Detection

[0083] The same mass of selenium-enriched Chlorella powder was used as the raw material for the experiment. Each group of experiments was repeated 3 times, and the average value was taken as the final test result. The specific test results are shown in the table below:

[0084] As can be seen from the table above...

[0085] In Comparative Example 1, no algal strain domestication was performed, and the organic selenium conversion rate was only 62.4%. However, in the three embodiments of this invention, the algal strains were domesticated through gradient selenium stress and ultraviolet mutagenesis, and the organic selenium conversion rate reached over 88%, with the highest reaching 91.7%. This demonstrates that the domestication method significantly improves the algal strains' selenium tolerance and selenium enrichment ability, thereby improving the conversion efficiency of inorganic selenium to organic selenium from the source.

[0086] Comparative Example 1 and Comparative Example 2 showed single-mode ultrasonic cell disruption and single-mode enzymatic cell disruption, with cell disruption rates of 61.2% and 78.5%, respectively. This invention uses a combination of enzymatic and ultrasonic cell disruption, with cell disruption rates exceeding 93%, resulting in more complete release of intracellular selenoproteins and directly leading to a significant increase in selenoprotein extraction yield. The yield of this example is almost double that of Comparative Example 1.

[0087] Comparative Example 1 uses a simplified impurity removal process, and Comparative Example 2 uses a simplified purification process, with the highest purity of selenoprotein being only 81.6%. This invention features a multi-step impurity removal process, including ethanol decolorization, fractional salting out, and low-temperature dialysis, which effectively removes impurities such as pigments, polysaccharides, and inorganic selenium. The purity of the finished product is higher than 91%, and the product quality is significantly improved.

[0088] Comparative Example 1 used hot air drying, which resulted in severe protein inactivation at high temperatures, with an activity retention rate of only 72.5%. In contrast, this invention uses mild acid and alkali treatment, low temperature treatment, and vacuum freeze drying throughout the process, maintaining a protein activity retention rate of over 96% and preserving the biological activity of selenoproteins to the greatest extent.

[0089] The three embodiments exhibit minimal fluctuations in various indicators, good adaptability of parameter ranges, and stable process flow. Combined with the advantages of high yield, high purity, and high activity, they fully meet the requirements for industrial-scale production.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting selenoproteins from selenium-rich Chlorella nucleatum, characterized in that, Includes the following steps: Step A, selenium-tolerant algal strain domestication: Select the original Chlorella proteoglycan strain, and use gradient selenium stress domestication combined with ultraviolet mutagenesis screening to obtain domesticated algal strains with high selenium tolerance and high accumulation capacity. Step B, Selenium-enriched culture: The domesticated algal strains are inoculated into a modified heterotrophic fermentation medium, and sodium selenite is added for selenium-enriched culture to obtain a selenium-enriched Chlorella protein-nucleated fermentation broth. Step C, Collection and Processing: Centrifuge, wash, and dry the selenium-enriched protein-nucleated Chlorella fermentation broth at low temperature, then pulverize and sieve to obtain selenium-enriched Chlorella dry powder, which is then stored in a sealed container at low temperature for later use. Step D, Composite Cell Wall Disruption Treatment: Selenium-enriched Chlorella powder is mixed with pre-cooled buffer to prepare an algal suspension. An enzymatic hydrolysis-ultrasonic composite cell wall disruption process is used to break down the algal cell walls step by step and release intracellular selenoproteins. Step E, Alkali Extraction: Adjust the algal suspension after cell wall disruption to an alkaline environment, stir and extract at a constant temperature to fully dissolve the selenoprotein, and centrifuge to obtain the crude selenoprotein supernatant. Step F, acid precipitation of protein: Adjust the pH of the crude extract supernatant to acidic at low temperature, let it stand at a constant temperature to precipitate, and collect the crude selenoprotein precipitate by centrifugation; Step G, Purification and Impurity Removal: A stepwise purification process is used to remove pigments, polysaccharides, inorganic selenium and small molecule impurities to obtain a purified selenium protein solution; Step H, Drying and Powdering: The refined selenium protein liquid is freeze-dried under low temperature and vacuum, then pulverized and sieved to obtain a high-purity selenium-enriched Chlorella selenium protein product.

2. The method for extracting selenoprotein from selenium-enriched Chlorella according to claim 1, characterized in that, The gradient selenium stress acclimatization in step A specifically involves: sequentially transferring the original Chlorella proteoglycans strain to basal culture media with sodium selenite concentration gradients of 100 mg / L, 300 mg / L, 500 mg / L, 800 mg / L, and 1500 mg / L, and acclimatizing in the dark for 5–7 days at each gradient to gradually improve the selenium tolerance of the algal strains; the ultraviolet mutagenesis screening specifically involves: spreading the acclimatized algal solution onto a plate, mutagenesis treatment at 30–40 cm under an ultraviolet lamp for 30–60 s, followed by dark incubation for 12 h to repair, and screening for single colonies with excellent growth, which are the highly selenium-tolerant acclimatized algal strains.

3. The method for extracting selenoprotein from selenium-rich Chlorella according to claim 1, characterized in that, In step B, the domesticated algal strain is inoculated into a modified heterotrophic fermentation medium with an inoculation volume fraction of 8%–12%, and cultured at a temperature of 25–30℃ and a pH of 5.5–6.0 for 2–3 days in the dark. Then, 2–4 mg / L of sodium selenite is added, the pH is adjusted to 6.0–7.0, the temperature is maintained at 25–30℃, and the culture continues for another 2–3 days to complete the biotransformation of inorganic selenium into organic selenoprotein. The modified heterotrophic fermentation medium formula is as follows: glucose 20 g / L, peptone 6 g / L, sodium nitrate 2 g / L, dipotassium hydrogen phosphate 0.08 g / L, magnesium sulfate heptahydrate 0.05 g / L, calcium chloride 0.01 g / L, sodium carbonate 0.04 g / L, citric acid 0.002 g / L, ferric citrate 0.003 g / L, trace element solution 1 g / L, sodium chloride 18 g / L, and the balance being distilled water.

4. The method for extracting selenoprotein from selenium-enriched Chlorella according to claim 1, characterized in that, In step C, the centrifugation speed is 5000-6000 rpm and the centrifugation time is 8-12 min. The product is washed 2-3 times with sterile distilled water. The drying temperature is 40-45℃ and the drying time is 8-12 h. After pulverization, the product is passed through an 80-100 mesh sieve.

5. The method for extracting selenoprotein from selenium-enriched Chlorella according to claim 1, characterized in that, Step D specifically includes: Step D1, Mixing: Mix the selenium-enriched Chlorella powder with a phosphate buffer solution of pH 7.0-7.4 at a ratio of 1:12 to 1:18 until homogeneous; Step D2, Enzymatic hydrolysis: Add a compound cell wall-breaking enzyme and hydrolyze at a constant temperature of 40-50℃ for 2-3 hours. The compound cell wall-breaking enzyme is a mixture of cellulase, pectinase, and snail enzyme. The total amount of the compound cell wall-breaking enzyme added is 1.5%-3.0% of the mass of the selenium-enriched Chlorella dry powder. Step D3, Ultrasound: Turn on ultrasound to facilitate cell wall disruption. The ultrasound power is 200-300W and the ultrasound frequency is 20-25kHz. The ultrasound works for 3 seconds, with a 5-second interval, and is cycled for 15-25 minutes to complete the composite cell wall disruption.

6. The method for extracting selenoprotein from selenium-rich Chlorella according to claim 1, characterized in that, In step E, the pH of the algal suspension is adjusted to 8.5–9.5 using 0.1 mol / L sodium hydroxide solution, and the suspension is stirred at room temperature for 1.5–2.5 h at a stirring speed of 150–200 rpm; the suspension is then centrifuged at 6000 rpm for 10–15 min.

7. The method for extracting selenoprotein from selenium-enriched Chlorella according to claim 1, characterized in that, In step F, the crude extract supernatant is cooled to 4-10°C, and the pH is slowly adjusted to 4.0-5.0 using glacial acetic acid. The mixture is then allowed to stand at low temperature for 2-4 hours to precipitate. After centrifugation at 6000 rpm for 10 minutes, the supernatant is discarded, and the grayish-white selenoprotein crude precipitate is collected.

8. The method for extracting selenoprotein from selenium-enriched Chlorella according to claim 1, characterized in that, The step-by-step purification process in step G specifically includes: Step G1, Decolorization: Add 85% to 95% anhydrous ethanol (by volume) to the crude selenoprotein precipitate at a ratio of 1:8 to 1:

12. Stir and decolorize at room temperature for 40 to 60 minutes, centrifuge at 6000 rpm for 10 minutes, discard the decolorization waste liquid, and repeat the decolorization process twice to remove phycobilichrome and lipid-soluble impurities. Step G2, Resolution and Removal: Resolute the decolorized protein precipitate with phosphate buffer, add ammonium sulfate for fractional salting out, control the saturation at 30% to 50%, let stand for 1 hour, and centrifuge to remove impurities such as proteins and polysaccharides. Step G3, dialysis desalting: The salted-out protein solution is placed into a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed at 4°C for 12-24 hours. The dialysis solution is changed every 4 hours to completely remove inorganic selenium, salt and small molecule impurities, and obtain a purified selenoprotein solution.

9. The method for extracting selenoprotein from selenium-enriched Chlorella according to claim 1, characterized in that, In step H, the vacuum degree is 0.08-0.1 MPa, the freezing temperature is -40 to -35℃, the vacuum freeze-drying time is 24-36 hours, and the powder is then passed through a 100-mesh sieve.

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