Preparation method of high-activity selenium protease with anti-oxidation and immunoregulation effects

Abstract

The invention discloses a preparation method of high-activity selenium protease with anti-oxidation and immunoregulation effects, and relates to the technical field of deep processing of protein and trace elements. The preparation method of the high-activity selenium protease comprises the following steps: preparing a fermentation culture medium; lactobacillus plantarum and bacillus licheniformis are activated and cultured, and are mixed and inoculated into a fermentation culture medium; fermenting and monitoring the pH value of a fermentation system in real time, and obtaining fermentation liquor after the fermentation is finished; and carrying out product treatment on the fermentation liquor by adopting a low-temperature sterilization and precise ultrafiltration coupling process to obtain the high-activity selenium protease. The high-activity selenium protease prepared by the preparation method disclosed by the invention has the effects of resisting oxidation and regulating immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of deep processing of proteins and trace elements, and particularly relates to a method for preparing a highly active selenoproteinase with antioxidant and immunomodulatory effects. Background Art

[0002] Amid the rapid development of the modern health industry, functional bioactive substances have become a focus of scientific research and market attention due to their key role in regulating human physiology. Selenium, an essential trace element for the human body, plays an irreplaceable role in the synthesis of selenoproteinases in physiological processes such as antioxidant and immune regulation. Research has shown that selenoproteinases can scavenge free radicals in the body, reduce oxidative stress damage, and enhance the activity of immune cells, playing a significant role in preventing various chronic diseases.

[0003] However, current selenoprotein production technology faces significant bottlenecks. Selenoproteins produced through traditional chemical synthesis or fermentation with a single bacterial strain have an average activity of only 60-80 U / mL, and after 30 days of storage at room temperature, the activity loss rate can reach 40%-50%. This makes it difficult to meet the demand for highly effective and stable functional ingredients in the food, health supplement, and pharmaceutical industries.

[0004] Therefore, the present invention provides a method for preparing a highly active selenoprotease with antioxidant and immunomodulatory effects, which is used to solve the technical problem in the traditional selenoprotease preparation method in the prior art that, due to the lack of a suitable selenium source and fermentation process, selenium cannot be effectively utilized by microorganisms, resulting in low activity and low yield of selenoprotease. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing highly active selenoproteases with antioxidant and immunomodulatory effects, which is used to solve the technical problem in the conventional selenoprotease preparation method in the prior art that, due to the lack of a suitable selenium source and fermentation process, selenium cannot be effectively utilized by microorganisms, resulting in low activity and low yield of selenoproteases.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing a highly active selenoproteinase with antioxidant and immunomodulatory effects comprises the following steps:

[0008] (1) preparing a fermentation medium, wherein the amount of the nano-selenium carrier added is 1.2-1.5 wt%;

[0009] (2) Activate and culture Lactobacillus plantarum and Bacillus licheniformis until the bacterial solution concentration reaches 1×10 8 -2×10 8 CFU / mL was mixed and inoculated into the fermentation medium;

[0010] (3) fermenting and monitoring the pH value of the fermentation system in real time, and obtaining a fermentation liquid after the fermentation is completed;

[0011] (4) The fermentation broth is treated by a low-temperature sterilization and precision ultrafiltration coupling process to obtain a highly active selenoprotease.

[0012] Furthermore, the fermentation medium in step (1) comprises, by mass ratio, 5-10% brown sugar, 3-5% glucose, 1-2% peptone, 0.3-0.5% vitamin C, 1.2-1.5% nano-selenium carrier, 0.3-0.5% superoxide dismutase, 0.5-0.8% zinc-enriched yeast, 1-1.5% protein, 0.3-0.5% glutathione, 0.1-0.3% coenzyme Q10, and the balance is water.

[0013] Furthermore, the superoxide dismutase activity is greater than or equal to 5000 U / mg.

[0014] The preparation method of the nano-selenium carrier comprises the following steps:

[0015] S1. Adding ethyl orthosilicate dropwise to a hexadecyltrimethylammonium bromide solution, stirring for reaction, washing by centrifugation, drying, calcining, and cooling to obtain mesoporous silica; ultrasonically dispersing the mesoporous silica in ethanol to obtain a mesoporous silica suspension;

[0016] S2. Dissolve FeCl3 and Na2S in water, add ascorbic acid, and introduce nitrogen for 1 hour to remove oxygen. Under nitrogen protection, stir and react to obtain a Fe3S4 suspension. Add Na2SeO3 and L-cysteine ​​to the Fe3S4 suspension, stir and react, and centrifuge to obtain nanoclusters. Disperse the nanoclusters in ethanol, mix with the MSN suspension, and stir and react to obtain a mesoporous silica support.

[0017] S3. Disperse the mesoporous silica load in 0.1 M phosphate buffer, add L-cysteine, stir to react, centrifuge to wash, and dry to obtain a nano-selenium carrier.

[0018] Preferably, the cetyltrimethylammonium bromide solution in S1 is prepared by dissolving 1 g of cetyltrimethylammonium bromide in 100 mL of deionized water, stirring at 40-42° C. until completely dissolved, adding 0.2 g of trimethylbenzene, continuing stirring for 20-25 minutes, adding 28 wt % ammonia water dropwise, and adjusting the pH to 10.5-11.

[0019] Preferably, the dropwise addition rate in S1 is 0.4-0.6 mL / min, the volume ratio of ethyl orthosilicate to hexadecyltrimethylammonium bromide solution is 1:(5-10); the stirring reaction speed is 280-300 rpm, the stirring reaction temperature is 80°C, the stirring reaction time is 6-8 h, the centrifugal speed is 8000-10000 rpm, the centrifugal time is 15 min, and the mixture is washed alternately with anhydrous ethanol and deionized water 3 times; the mixture is dried in a vacuum drying oven at 60°C for 12 hours; during calcination, the muffle furnace is heated to 540-560°C at 1-1.2°C / min and maintained for 5 hours; the pore size of the mesoporous silica is 4-6 nm; and the amount ratio of mesoporous silica to ethanol is (0.1-0.5) g:50 mL.

[0020] Preferably, the dosage ratio of FeCl3, Na2S, Na2SeO3, L-cysteine, ascorbic acid and water in S2 is 1.5mol:2mol:0.4-0.5mol:2mol:1g:100mL, the stirring speed is 250-300rpm, the stirring temperature is 75-80℃, and the stirring reaction time is 2-3h.

[0021] Preferably, the pH of the phosphate buffer in S3 is 7.4, the concentration of L-cysteine ​​is 5 mM, the stirring reaction time is 8 h, the centrifugal speed is 6000-8000 rpm, the centrifugal time is 10-15 min, and the free ligand is removed by washing with deionized water 3-4 times.

[0022] Furthermore, in step (2), the volume ratio of Lactobacillus plantarum to Bacillus licheniformis is (2-3):1, the inoculation is performed at an inoculation amount of 2-5vt%, and the pH during inoculation is controlled at 6.8-7.2.

[0023] Furthermore, in step (3), the fermentation temperature is 35-37° C., and the fermentation time is 36-48 h. During fermentation, potassium citrate solution and citric acid solution are used to maintain the pH of the fermentation liquid at 6.8-7.2. When the pH is close to 6.8, potassium citrate solution is added; when the pH is close to 7.2, citric acid solution is added; and the stirring speed is 150-200 rpm.

[0024] Furthermore, in step (4), low-temperature sterilization is performed by placing the fermentation broth at 0-4°C and performing sterile filtration using a 0.22 μm filter membrane to obtain a sterilized fermentation broth; precision ultrafiltration is performed by filtering the sterilized fermentation broth through a 5-10 kDa ultrafiltration membrane to remove small molecular impurities and large molecular protein polymers in the fermentation broth, collecting the retentate, dialyzing it three times with deionized water, concentrating and freeze-drying it, adding 5 wt% mannitol and 2 wt% trehalose, pre-freezing it at -80°C for 4-6 h, and freeze-drying it for 46-48 h to obtain a highly active selenoprotease with antioxidant and immunomodulatory effects.

[0025] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0026] 1. The present invention utilizes the different metabolic characteristics of Lactobacillus plantarum and Bacillus licheniformis. Lactobacillus plantarum can produce organic acids such as lactic acid, regulate the pH of the fermentation environment, inhibit the growth of harmful microorganisms, and synthesize some beneficial metabolites. Bacillus licheniformis has a strong decomposition ability and can secrete a variety of enzymes to decompose macromolecules into small molecules, providing Lactobacillus plantarum with more available nutrients. The mixed fermentation method is adopted to jointly promote the fermentation process and improve the yield and quality of selenoprotease.

[0027] 2. The present invention provides selenium to bacteria through nano-selenium carriers, participates in the formation process of selenoproteinase, improves the activity and content of the enzyme, and enhances its antioxidant and immunomodulatory effects. Among them, mesoporous silica, as the basic material of nano-selenium carriers, has a regular mesoporous structure with a pore size of 4-6nm. This mesoporous structure provides a large specific surface area and pore volume, which can load more nanoclusters and bioactive ingredients, and provides a good physical space for the loading of selenium and subsequent interaction with bacteria. Moreover, the loading of Fe3S4 and nanoclusters on mesoporous silica increases the functional properties of nano-selenium carriers. Fe3S4 has magnetic and catalytic activity, participates in metabolic processes and enhances the interaction between carriers and biomolecules; the selenium contained in nanoclusters is a key component of selenoproteinase. Through effective loading methods, selenium can be better utilized by bacteria, thereby improving the synthesis efficiency of selenoproteinase. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 This is a process flow chart for preparing highly active selenoprotease in an embodiment of the present invention;

[0030] Figure 2 This is a line graph showing the activity determination of selenoproteases in Examples 1-3 and Comparative Examples 1-4 of the present invention;

[0031] Figure 3 This is a line graph showing the selenium content of Examples 1-3 and Comparative Examples 1-4 of the present invention. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] Example 1:

[0034] This embodiment discloses a method for preparing a nano-selenium carrier, comprising the following steps:

[0035] S1. Dissolve 1 g of hexadecyltrimethylammonium bromide in 100 mL of deionized water, stir at 40°C until completely dissolved, add 0.2 g of trimethylbenzene, continue stirring for 20 min, add 28 wt% ammonia water dropwise, adjust the pH to 10.5, and obtain a hexadecyltrimethylammonium bromide solution; add 10 mL of ethyl orthosilicate dropwise to 50 mL of the hexadecyltrimethylammonium bromide solution at a dropping speed of 0.4 mL / min, and stir the reaction at a stirring speed of 280 rpm. The reaction temperature was 80°C, the stirring reaction time was 6 hours, the mixture was centrifuged at a speed of 8000 rpm for 15 minutes, and the mixture was washed alternately with anhydrous ethanol and deionized water three times; the mixture was dried in a vacuum drying oven at 60°C for 12 hours, and calcined. During the calcination, the temperature in the muffle furnace was raised to 540°C at a rate of 1°C / min and maintained for 5 hours; the mixture was cooled to obtain mesoporous silica with a pore size of 4 nm; 0.1 g of the mesoporous silica was ultrasonically dispersed in 50 mL of ethanol to obtain a mesoporous silica suspension;

[0036] S2. Dissolve 1.5 mol FeCl3 and 2 mol Na2S in 100 mL water, add 1 g ascorbic acid, and introduce nitrogen for 1 hour to remove oxygen. Under nitrogen protection, stir the mixture at a speed of 250 rpm, a temperature of 75°C, and a stirring reaction time of 2 hours to obtain a Fe3S4 suspension; add 0.4 mol Na2SeO3 and 2 mol L-cysteine ​​to the Fe3S4 suspension, stir the mixture at a speed of 300 rpm, and centrifuge to obtain nanoclusters; disperse the nanoclusters in ethanol, mix the mixture with the mesoporous silica suspension, and stir the mixture to obtain a mesoporous silica support;

[0037] S3. Disperse the mesoporous silica carrier in 0.1M phosphate buffer with a pH of 7.4, add 5mM L-cysteine, stir the reaction, and the reaction time is 8 hours. Centrifuge and wash at a speed of 6000 rpm for 10 minutes. Wash with deionized water three times to remove free ligands, and dry to obtain a nano-selenium carrier.

[0038] This example discloses a method for preparing a highly active selenoproteinase with antioxidant and immunomodulatory effects, comprising the following steps:

[0039] (1) preparing a fermentation medium, wherein the fermentation medium comprises, by mass ratio, 5% brown sugar, 3% glucose, 1% peptone, 0.3% vitamin C, 1.2% nano-selenium carrier, 0.3% superoxide dismutase, 0.5% zinc-enriched yeast, 1% protein, 0.3% glutathione, 0.1% coenzyme Q10, and the balance being water;

[0040] (2) Lactobacillus plantarum and Bacillus licheniformis were activated and cultured, with the volume ratio of Lactobacillus plantarum to Bacillus licheniformis being 3:1. When the bacterial solution concentration reached 1×10 8 CFU / mL were mixed and inoculated into the fermentation medium at an inoculum size of 2vt%, and the pH was controlled at 6.8;

[0041] (3) Fermentation and real-time monitoring of the fermentation system pH value of 6.8, fermentation temperature of 35° C., fermentation time of 36 h, and fermentation completion to obtain fermentation liquid;

[0042] (4) The fermentation broth was treated by a low-temperature sterilization and precision ultrafiltration coupling process. The low-temperature sterilization process was to place the fermentation broth at 0°C and use a 0.22 μm filter membrane for sterilization filtration to obtain a sterilized fermentation broth; the precision ultrafiltration process was to filter the sterilized fermentation broth through a 5 kDa ultrafiltration membrane to remove small molecular impurities and large molecular protein polymers in the fermentation broth, collect the retentate, dialyze 3 times with deionized water, concentrate and freeze-dry, add 5 wt% mannitol and 2 wt% trehalose, pre-freeze at -80°C for 4 h, and freeze-dry for 46 h to obtain a highly active selenoproteinase with antioxidant and immunomodulatory effects.

[0043] Example 2:

[0044] This embodiment discloses a method for preparing a nano-selenium carrier, comprising the following steps:

[0045] S1, 1g of hexadecyltrimethylammonium bromide was dissolved in 100mL of deionized water, stirred at 41°C until completely dissolved, 0.2g of trimethylbenzene was added, stirring was continued for 23min, 28wt% ammonia water was added dropwise, and the pH was adjusted to 10.8 to obtain a hexadecyltrimethylammonium bromide solution; 10mL of ethyl orthosilicate was added dropwise to 80mL of the hexadecyltrimethylammonium bromide solution at a dropping speed of 0.5mL / min, and the reaction was stirred at a speed of 290rpm. The stirring reaction temperature was 400℃. The mixture was stirred at 80°C for 7 hours, washed by centrifugation at a speed of 9000 rpm for 15 minutes, and washed three times alternately with anhydrous ethanol and deionized water; dried in a vacuum drying oven at 60°C for 12 hours, and calcined. During the calcination, the temperature in the muffle furnace was raised to 550°C at a rate of 1.1°C / min and maintained for 5 hours; cooled to obtain mesoporous silica with a pore size of 5 nm; and 0.1-0.5 g of the mesoporous silica was ultrasonically dispersed in 50 mL of ethanol to obtain a mesoporous silica suspension.

[0046] S2. Dissolve 1.5 mol FeCl3 and 2 mol Na2S in 100 mL water, add 1 g ascorbic acid, introduce nitrogen for 1 hour to remove oxygen, and react with stirring under nitrogen protection at a speed of 280 rpm and a temperature of 78°C for 2-3 hours to obtain a Fe3S4 suspension; add 0.45 mol Na2SeO3 and 2 mol L-cysteine ​​to the Fe3S4 suspension, react with stirring at a speed of 300 rpm, and centrifuge to obtain nanoclusters; disperse the nanoclusters in ethanol, mix with the mesoporous silica suspension, and react with stirring to obtain a mesoporous silica support;

[0047] S3. Disperse the mesoporous silica carrier in 0.1M phosphate buffer with a pH of 7.4, add 5mM L-cysteine, stir the reaction, and the reaction time is 8 hours. Centrifuge and wash at a speed of 7000 rpm and a time of 13 minutes. Wash with deionized water three times to remove free ligands, and dry to obtain a nano-selenium carrier.

[0048] This example discloses a method for preparing a highly active selenoproteinase with antioxidant and immunomodulatory effects, comprising the following steps:

[0049] (1) preparing a fermentation medium, wherein the fermentation medium comprises, by mass ratio, 8% brown sugar, 4% glucose, 1.5% peptone, 0.4% vitamin C, 1.4% nano-selenium carrier, 0.4% superoxide dismutase, 0.7% zinc-enriched yeast, 1.3% protein, 0.4% glutathione, 0.2% coenzyme Q10, and the balance being water;

[0050] (2) Lactobacillus plantarum and Bacillus licheniformis were activated and cultured, with a volume ratio of Lactobacillus plantarum to Bacillus licheniformis of 2.5:1. When the bacterial solution concentration reached 1.5×10 8 CFU / mL were mixed and inoculated into the fermentation medium at an inoculum size of 4vt%, and the pH was controlled at 7;

[0051] (3) fermenting and real-time monitoring the pH value of the fermentation system to 7, the fermentation temperature to 36° C., the fermentation time to 42 h, and the fermentation to obtain a fermentation liquid;

[0052] (4) The fermentation broth was treated by a low-temperature sterilization and precision ultrafiltration coupling process. The low-temperature sterilization process was to place the fermentation broth at 2°C and sterilize and filter it with a 0.22 μm filter membrane to obtain a sterilized fermentation broth. The precision ultrafiltration process was to filter the sterilized fermentation broth through an 8 kDa ultrafiltration membrane to remove small molecular impurities and large molecular protein polymers in the fermentation broth, collect the retentate, dialyze it three times with deionized water, concentrate and freeze-dry it, add 5 wt% mannitol and 2 wt% trehalose, pre-freeze it at -80°C for 5 h, and freeze-dry it for 47 h to obtain a highly active selenoproteinase with antioxidant and immunomodulatory effects.

[0053] Example 3:

[0054] This embodiment discloses a method for preparing a nano-selenium carrier, comprising the following steps:

[0055] S1. Dissolve 1 g of hexadecyltrimethylammonium bromide in 100 mL of deionized water, stir at 42° C. until completely dissolved, add 0.2 g of trimethylbenzene, continue stirring for 25 min, add 28 wt % ammonia water dropwise, adjust the pH to 10.5-11, and obtain a hexadecyltrimethylammonium bromide solution; add 10 mL of ethyl orthosilicate dropwise to the 100 mL hexadecyltrimethylammonium bromide solution at a dropping speed of 0.6 mL / min, stir the reaction at a stirring speed of 300 rpm, and stir for 5 minutes. The stirring reaction temperature was 80°C, the stirring reaction time was 8 hours, the centrifugal washing was performed at a speed of 10,000 rpm, the centrifugal time was 15 minutes, and the mixture was washed alternately with anhydrous ethanol and deionized water three times; the mixture was dried in a vacuum drying oven at 60°C for 12 hours, and calcined. During the calcination, the temperature of the muffle furnace was increased to 560°C at a rate of 1.2°C / min and maintained for 5 hours; the mixture was cooled to obtain mesoporous silica with a pore size of 6 nm; 0.5 g of the mesoporous silica was ultrasonically dispersed in 50 mL of ethanol to obtain a mesoporous silica suspension;

[0056] S2. Dissolve 1.5 mol FeCl3 and 2 mol Na2S in 100 mL water, add 1 g ascorbic acid, introduce nitrogen for 1 hour to remove oxygen, and stir under nitrogen protection at a speed of 300 rpm, a temperature of 80°C, and a stirring reaction time of 3 hours to obtain a Fe3S4 suspension; add 0.5 mol Na2SeO3 and 2 mol L-cysteine ​​to the Fe3S4 suspension, stir and react at a speed of 300 rpm, and centrifuge to obtain nanoclusters; disperse the nanoclusters in ethanol, mix with the mesoporous silica suspension, and stir to obtain a mesoporous silica support;

[0057] S3. Disperse the mesoporous silica carrier in 0.1M phosphate buffer with a pH of 7.4, add 5mM L-cysteine, stir the reaction, and the reaction time is 8 hours. Centrifuge and wash at a speed of 8000 rpm and a time of 15 minutes. Wash with deionized water 4 times to remove free ligands, and dry to obtain a nano-selenium carrier.

[0058] This example discloses a method for preparing a highly active selenoproteinase with antioxidant and immunomodulatory effects, comprising the following steps:

[0059] (1) preparing a fermentation medium, wherein the fermentation medium comprises, by mass ratio, 10% brown sugar, 5% glucose, 2% peptone, 0.5% vitamin C, 1.5% nano-selenium carrier, 0.5% superoxide dismutase, 0.8% zinc-enriched yeast, 1.5% protein, 0.5% glutathione, 0.1-0.3% coenzyme Q10, and the balance being water;

[0060] (2) Lactobacillus plantarum and Bacillus licheniformis were activated and cultured, with the volume ratio of Lactobacillus plantarum to Bacillus licheniformis being 2:1. When the bacterial solution concentration reached 2×10 8 CFU / mL was mixed and inoculated into the fermentation medium at an inoculum size of 2-5vt%, and the pH was controlled at 7.2;

[0061] (3) fermenting and real-time monitoring the pH value of the fermentation system to 7.2, the fermentation temperature to 37° C., the fermentation time to 48 h, and obtaining the fermentation liquid after the fermentation is completed;

[0062] (4) The fermentation broth was treated by a low-temperature sterilization and precision ultrafiltration coupling process. The low-temperature sterilization process was to place the fermentation broth at 4°C and sterilize and filter it with a 0.22 μm filter membrane to obtain a sterilized fermentation broth. The precision ultrafiltration process was to filter the sterilized fermentation broth through a 10 kDa ultrafiltration membrane to remove small molecular impurities and large molecular protein polymers in the fermentation broth, collect the retentate, dialyze it three times with deionized water, concentrate and freeze-dry it, add 5 wt% mannitol and 2 wt% trehalose, pre-freeze it at -80°C for 6 h, and freeze-dry it for 48 h to obtain a highly active selenoproteinase with antioxidant and immunomodulatory effects.

[0063] Comparative Example 1:

[0064] This example discloses a method for preparing a highly active selenoproteinase with antioxidant and immunomodulatory effects, comprising the following steps:

[0065] (1) preparing a fermentation medium, wherein the fermentation medium comprises 10% brown sugar, 5% glucose, 2% peptone, 0.5% vitamin C, 1.5% nano-selenium carrier, 0.5% superoxide dismutase, 0.8% zinc-enriched yeast, 1.5% protein, 0.5% glutathione, 0.1-0.3% coenzyme Q10, and the balance is water;

[0066] (2) Lactobacillus plantarum and Bacillus licheniformis were activated and cultured, with the volume ratio of Lactobacillus plantarum to Bacillus licheniformis being 1:2. When the bacterial solution concentration reached 2×10 8 CFU / mL was mixed and inoculated into the fermentation medium at an inoculum size of 2-5vt%, and the pH was controlled at 7.2;

[0067] (3) fermenting and real-time monitoring the pH value of the fermentation system to be 6.8-7.2, the fermentation temperature to be 35-37° C., the fermentation time to be 36-48 h, and the fermentation to be completed to obtain the fermentation liquid;

[0068] (4) The fermentation broth was treated by a low-temperature sterilization and precision ultrafiltration coupling process. The low-temperature sterilization process was to place the fermentation broth at 4°C and sterilize and filter it with a 0.22 μm filter membrane to obtain a sterilized fermentation broth; the precision ultrafiltration process was to filter the sterilized fermentation broth through a 10 kDa ultrafiltration membrane to remove small molecular impurities and large molecular protein polymers in the fermentation broth, collect the retentate, dialyze it three times with deionized water, concentrate and freeze-dry it, add 5 wt% mannitol and 2 wt% trehalose, pre-freeze it at -80°C for 4 hours, and freeze-dry it for 48 hours to obtain a highly active selenoproteinase with antioxidant and immunomodulatory effects.

[0069] Comparative Example 1 Compared with Example 3, in the preparation process of high-activity selenoprotease in Comparative Example 1, the volume ratio of Lactobacillus plantarum to Bacillus licheniformis was 1:2, and other conditions remained unchanged.

[0070] Comparative Example 2:

[0071] Comparative Example 2 Compared with Example 3, in the preparation process of high-activity selenoprotease in Comparative Example 2, the nano-selenium carrier was replaced with sodium selenite, and other conditions remained unchanged.

[0072] Comparative Example 3:

[0073] Comparative Example 3 Compared with Example 3, in the preparation process of high-activity selenoprotease in Comparative Example 3, Lactobacillus plantarum was not added, and other conditions remained unchanged.

[0074] Comparative Example 4:

[0075] Comparative Example 4 Compared with Example 3, in the preparation process of high-activity selenoprotease in Comparative Example 4, Bacillus licheniformis was not added, and other conditions remained unchanged.

[0076] Experimental example:

[0077] The selenoproteases prepared in Examples 1-3 and Comparative Examples 1-4 were tested for selenoproteases activity and selenium content according to GB 5009.93-2017. The results are shown in Table 1.

[0078] Table 1 Selenoprotease activity and selenium content

[0079]

[0080] According to the measurement results in Table 1, it can be seen that the selenoprotease activity and selenium content of the high-activity selenoprotease prepared in Examples 1-3 are higher than those in Comparative Examples 1-4. By comparing Examples 1-3 with Comparative Examples 1-4, it can be seen that the inoculation ratio of Lactobacillus plantarum to Bacillus licheniformis and the nano-selenium carrier are helpful to improve the selenoprotease activity and selenium content.

[0081] The antioxidant activity of the highly active selenoproteases prepared in Examples 1-3 and Comparative Examples 1-4 was determined according to GB / T 33410-2016 "Superoxide Anion Radical Scavenging Rate". The results are shown in Table 2.

[0082] According to GB / T 16886.5-2017 "MTT method", the highly active selenoproteases prepared in Examples 1-3 and Comparative Examples 1-4 were used to detect the proliferation rate of mouse spleen lymphocytes for immunomodulatory activity determination. The results of the determination are shown in Table 2.

[0083] Among them, the superoxide anion free radical scavenging rate reflects the ability of selenoproteinase to scaveng superoxide anion free radicals (O2 - ·) ability, the higher the value, the stronger the antioxidant activity, O2 - It is the main component of reactive oxygen species (ROS). Excessive accumulation leads to oxidative damage. Its high clearance rate indicates that the protease has significant antioxidant protection. The MTT assay was used to determine the efficiency of selenoprotease in promoting the proliferation of mouse spleen lymphocytes. A higher lymphocyte proliferation rate indicates a stronger immunomodulatory activity.

[0084] Table 2 Antioxidant and immunomodulatory activities of selenoproteinases

[0085] project Superoxide anion free radical scavenging rate (%) Lymphocyte proliferation rate (%) Example 1 82.2 118.5 Example 2 82.5 118.7 Example 3 82.6 119.0 Comparative Example 1 73.5 85.2 Comparative Example 2 52.6 69.3 Comparative Example 3 41.2 60.1 Comparative Example 4 41.5 60.3

[0086] According to the measurement results in Table 2, it can be seen that the superoxide anion radical scavenging rate and lymphocyte proliferation rate of the highly active selenoproteases prepared in Examples 1-3 are higher than those in Comparative Examples 1-4. By comparing Examples 1-3 with Comparative Examples 1-4, it can be seen that the inoculation ratio of Lactobacillus plantarum to Bacillus licheniformis and the nano-selenium carrier help to improve the antioxidant activity and immunomodulatory activity of selenoproteases.

[0087] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

[0088] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to specific embodiments. Obviously, many modifications and variations are possible based on the contents of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing a highly active selenoproteinase with antioxidant and immunomodulatory effects, characterized in that: The following steps are involved: (1) preparing a fermentation medium, wherein the amount of the nano-selenium carrier added is 1.2-1.5 wt%; (2) Activate and culture Lactobacillus plantarum and Bacillus licheniformis until the bacterial solution concentration reaches 1×10 8 -2×10 8 CFU / mL were mixed and inoculated into the fermentation medium; (3) Fermentation and real-time monitoring of the pH value of the fermentation system to 6.8-7.2, and after 36-48 hours, obtaining the fermentation liquid; (4) The fermentation broth is treated by a low-temperature sterilization and ultrafiltration coupling process to obtain a highly active selenoprotease.

2. The method for preparing a highly active selenoprotease having antioxidant and immunomodulatory effects according to claim 1, wherein: In step (1), the fermentation medium comprises, by mass ratio, 5-10% brown sugar, 3-5% glucose, 1-2% peptone, 0.3-0.5% vitamin C, 1.2-1.5% nano-selenium carrier, 0.3-0.5% superoxide dismutase, 0.5-0.8% zinc-enriched yeast, 1-1.5% protein, 0.3-0.5% glutathione, 0.1-0.3% coenzyme Q10, and the balance is water.

3. The method for preparing a highly active selenoproteinase having antioxidant and immunomodulatory effects according to claim 2, wherein: The preparation method of the nano-selenium carrier comprises the following steps: S1. Adding ethyl orthosilicate dropwise to a hexadecyltrimethylammonium bromide solution, stirring for reaction, washing by centrifugation, drying, calcining, and cooling to obtain mesoporous silica; ultrasonically dispersing the mesoporous silica in ethanol to obtain a mesoporous silica suspension; S2, dissolving FeCl3 and Na2S powders in water, adding ascorbic acid, and stirring under nitrogen to react to obtain a Fe3S4 suspension; adding Na2SeO3 powder and L-cysteine ​​to the Fe3S4 suspension, stirring to react, and centrifuging to obtain nanoclusters; dispersing the nanoclusters in ethanol, mixing with the mesoporous silica suspension, and stirring to react to obtain a mesoporous silica support; S3. Disperse the mesoporous silica load in 0.1 M phosphate buffer, add L-cysteine, stir to react, centrifuge to wash, and dry to obtain a nano-selenium carrier.

4. The method for preparing a highly active selenoprotease having antioxidant and immunomodulatory effects according to claim 3, wherein: The cetyltrimethylammonium bromide solution in S1 is prepared by dissolving 1 g of cetyltrimethylammonium bromide in 100 mL of deionized water, stirring at 40-42° C. until completely dissolved, adding 0.2 g of trimethylbenzene, continuing stirring for 20-25 minutes, adding 28 wt % ammonia water dropwise, and adjusting the pH to 10.5-11.

5. The method for preparing a highly active selenoproteinase having antioxidant and immunomodulatory effects according to claim 3, wherein: The volume ratio of ethyl orthosilicate to hexadecyltrimethylammonium bromide solution in S1 is 1:(5-10); the stirring reaction speed is 280-300 rpm; during calcination, the muffle furnace is heated to 540-560°C at 1-1.2°C / min and maintained for 5 hours; the pore size of the mesoporous silica is 4-6 nm; and the dosage ratio of mesoporous silica to ethanol is (0.1-0.5) g:50 mL.

6. The method for preparing a highly active selenoproteinase having antioxidant and immunomodulatory effects according to claim 3, wherein: The dosage ratio of FeCl3, Na2S, Na2SeO3, L-cysteine, ascorbic acid and water in S2 is 1.5mol:2mol:0.4-0.5mol:2mol:1g:100mL.

7. The method for preparing a highly active selenoprotease with antioxidant and immunomodulatory effects according to claim 3, wherein: The pH of the phosphate buffer in S3 was 7.4, and the concentration of L-cysteine ​​was 5 mM.

8. The method for preparing a highly active selenoprotease with antioxidant and immunomodulatory effects according to claim 1, wherein: In step (2), the volume ratio of Lactobacillus plantarum to Bacillus licheniformis is (2-3):1, and the inoculation is performed at an inoculation amount of 2-5vt%, and the pH during inoculation is controlled at 6.8-7.

2.

9. The method for preparing a highly active selenoprotease with antioxidant and immunomodulatory effects according to claim 1, wherein: In step (3), potassium citrate solution and citric acid solution are used to maintain the pH of the fermentation broth at 6.8-7.

2.

10. The method for preparing a highly active selenoprotease with antioxidant and immunomodulatory effects according to claim 1, wherein: In step (4), low-temperature sterilization is to place the fermentation liquid at 0-4°C and filter it with a 0.22 μm filter membrane to obtain a sterilized fermentation liquid; precision ultrafiltration is to filter the sterilized fermentation liquid through a 5-10 kDa ultrafiltration membrane.

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