Preparation and use of a highly active and safe selenoprotein standard sample derived from Cardamine violifolia

Abstract

The purpose of this study is to produce and use a highly active and safe standard sample of selenoprotein derived from Cardamine violifolia. In the method of the present invention, Cardamine violifolia powder is hydrolyzed with cellulase, and after completion of enzymatic decomposition, the mixture is centrifuged to obtain a supernatant. Then, a complex enzyme is added to the supernatant to continue the enzymatic decomposition process, and electrodialysis is simultaneously performed. The resulting reaction solution is concentrated and dried after inactivating the enzyme to obtain a selenoprotein standard sample. The selenoprotein standard sample produced by the present invention has the advantages of extremely low heavy metal content, low ash content, high protein content, high total selenium content, organic selenium content reaching 99.9% or more, high biological activity, and high safety. Among them, organic selenium is mainly composed of selenocysteine / selenocystine, which accounts for more than 90% of the total selenium content, and is expected to make up for the shortage of selenoprotein standard samples in the market.

Description

[Technical field]

[0001] The present invention relates to the technical field of selenoproteins, and more particularly to a process for producing a standard sample of Cardamine violifolia-derived selenoproteins with high biological activity, high safety and low heavy metal content, and the use of the obtained standard sample of Cardamine violifolia-derived selenoproteins in the production of foods and medicines. [Background technology]

[0002] "GB1903.28-2018 National Standard for Food Safety Food Enhancer Selenoprotein" discloses a selenoprotein standard product, which specifies that the source of selenoprotein is a food enhancer rich in selenomethionine, which is purified from edible plants such as soybeans with high selenium content through the processes of degreasing, water extraction, ethanol precipitation, and drying. However, due to the problem that the selenium content of soybean raw materials is generally low and the purification technology is insufficient, there are no selenoprotein products that meet the standard on the current market, and even the production of standard samples for laboratory use is difficult, which presents a major obstacle to industrial use. In addition, as research has shown, selenocysteine / selenocystine is safer than selenomethionine, and selenocysteine ​​has already been defined in academia as the 21st essential amino acid of the human body, so selenoproteins with high biological activity, high safety, and high selenocysteine / selenocystine content have important application value.

[0003] Plants rich in selenium, such as broccoli, cabbage, Cardamine violifolia, and rice, usually also contain a lot of heavy metal contaminants. These heavy metals are mainly cadmium, lead, and arsenic, because the heavy metal content in the selenium-rich soil in which selenium-rich plants are grown is usually high, and selenium and cadmium are associated, so the plants are rich in selenium and also rich in a certain amount of heavy metals, which results in the heavy metals in the plants exceeding the standard value, which further poses a risk to food safety. Cardamine violifolia, in particular, is a plant with a very strong ability to accumulate selenium, and the form of organic selenium is mainly selenocysteine ​​(calculated as selenocystine). However, currently, Cardamine violifolia is often eaten as it is, dried and crushed to be used as food raw materials, or simply extracted or extracted with organic solvents, and the risk of food contamination by heavy metals is high when the soil has a high background value of heavy metals. In addition, most heavy metals in plants form complexes with protein structures at the active center of enzymes and peptides, for example, by binding with plant chelating peptides to improve the plant's resistance to heavy metals. Therefore, water extraction and simple organic solvent extraction cannot effectively destroy the bonds between heavy metals and chelating peptides, and cannot effectively remove heavy metals.

[0004] Currently, organic solvents, strong acids, strong alkalis, ethanol, etc. are generally used to extract organic selenoproteins from plants, which not only easily destroy the quaternary structure of selenoproteins and reduce the stability of functional components, leading to various degrees of inactivation, but also generate large amounts of wastewater and waste liquid, causing serious environmental pollution and posing production safety risks.On the other hand, conventional water extraction methods contain high amounts of components such as heavy metals and ash, posing food safety risks, and the form of the separated selenium components is unclear, so their efficacy, activity, and safety cannot be guaranteed. Summary of the Invention [Problem to be solved by the invention]

[0005] In response to the problems of the prior art, the present invention provides a process for producing a selenoprotein standard sample with low heavy metal content, high biological activity, high safety and high selenocysteine / selenocystine content by using Cardamine violifolia as raw material. In the selenoprotein standard sample produced by this process, the selenocysteine / selenocystine content can account for more than 90% of the total selenium content, and the total selenium content, heavy metal content, protein content and characteristic organic selenium forms all meet or significantly exceed the requirements of the national standard for selenoprotein. [Means for solving the problem]

[0006] The technical solutions of the present invention are specifically as follows:

[0007] A method for producing a highly active and safe standard sample of selenoprotein derived from Cardamine violifolia, specifically, Step 1 is to hydrolyze Cardamine biolifolia powder with cellulase, and after the enzymatic hydrolysis is completed, the enzyme is inactivated and centrifuged to obtain the supernatant; Step 2: adding a complex enzyme consisting of alkaline protease, trypsin, papain, protease K, and protease XIV to the supernatant to continue the enzymatic decomposition treatment, and simultaneously carrying out electrodialysis to dissociate heavy metals complexed with proteins, and then removing heavy metal ions and inorganic selenium salt ions by electrodialysis, followed by inactivating the enzymes; and step 3 of concentrating and drying the reaction solution obtained in step 2 to obtain a selenoprotein standard sample.

[0008] Furthermore, in the above technical solution, the amount of cellulase added in step 1 is 0.1-10 wt% of the Cardamine violifolia powder, and the enzymatic decomposition by cellulase is performed at a temperature of 30-50°C for 1-4 hours.

[0009] Furthermore, in the above technical solution, the parameters of the centrifugation in step 1 are 1000-10000 r / min and 5-60 min, and part of the large insoluble matter remaining after cellulase decomposition is removed by centrifugation.

[0010] Furthermore, in the above technical solution, the amount of the complex enzymes added in step 2 is 0.1-10 wt% of the Cardamine violifolia powder, the temperature of the enzymatic hydrolysis by the complex enzymes is 30-50°C, and further, the mass ratio of alkaline protease, trypsin, papain, protease K and protease XIV in the complex enzymes is 1:1:1:1:1.

[0011] Furthermore, in the above technical solution, the parameters of the electrodialysis in step 2 are: voltage 15-100V, current ≦10A, electrode water flow rate 1-100L / h, fresh water and concentrated water flow rates 1-100L / h, fresh water and concentrated water volume ratio is 1:1, and the electrode water uses 0.1-10wt% sodium sulfate solution.

[0012] Furthermore, the treatment time for enzymatic decomposition and electrodialysis in step 2 is 0.5 to 8 hours.

[0013] Furthermore, in the above technical solutions, the temperature of the enzyme inactivation treatment is 85-95°C.

[0014] Furthermore, in the above technical solutions, the drying method in step 3 is spray drying or freeze drying. Effect of the Invention

[0015] The beneficial effects of the present invention are as follows: 1) First, cellulase is used to decompose the fiber bound to the protein in Cardamine violifolia, releasing the protein rich in organic selenium; then, the complex enzyme targets the high molecular weight protein and cuts it into amino acids; the complex enzyme of the present invention can efficiently and maximally selectively dissociate selenocysteine / selenocystine; the complex enzyme can dissociate the heavy metals complexed with the protein during the enzymatic decomposition and further turn them into free heavy metal ions; and, combined with electrodialysis technology, remove the heavy metal ions and inorganic selenium without affecting the stability of the selenoprotein characterized by highly active selenocysteine / selenocystine. 2) In the present invention, the enzymatic hydrolysis conditions are mild, the enzymatic hydrolysis time is short, and no strong acid, strong alkali, or organic solvent is used, so that the steady-state conversion of the organoselenium form of selenocysteine / selenocystine can be maximized and protected, and the physiological activity can be maintained. 3) The present invention found that the efficient and selective dissociation of selenoproteins rich in selenocysteine / selenocystine could be promoted by conjugated enzymes through electrodialysis. 4) In the standard sample of selenoprotein derived from Cardamine violifolia prepared in the present invention, the selenium form is mainly selenocysteine / selenocystine, and its content can reach more than 90% of the total selenium content. Selenocysteine / selenocystine is safer than selenomethionine. In the present invention, its high activity and high safety were further verified through animal tests and applications. 5) In the standard sample of selenoprotein derived from Cardamine violifolia prepared by the present invention, the total selenium content is 1500-5000mg / kg, the proportion of organic selenium content in the total selenium content can reach 99.9% or more, and the organic selenium content is far higher than the organic selenium content of 80% or more required by "DBS42 / 002-2022 Requirements for Selenium Content in Organic Selenium-rich Foods". Therefore, the safety risk caused by inorganic selenium residue can be almost eliminated, so the safety is higher. The protein content in selenoprotein is 60% or more, heavy metals such as lead, arsenic, mercury, and cadmium in the extract are all undetectable, and the ash content is 2% or less, so its safety is also high. From the above, each parameter of the standard sample of selenoprotein derived from Cardamine violifolia obtained by the present invention can meet or significantly exceed the requirements of the national standard for selenoprotein. Please refer to the following table for details. JPEG0007608004000001.jpg87170

[0016] From the above table, it can be expected that the selenoprotein standard sample derived from Cardamine biofolii produced in the present invention can make up for the shortage of selenoprotein standard products on the market. [Brief description of the drawings]

[0017] [Figure 1] 1 is a graph showing the change in serum glutathione peroxidase activity after a single administration of a selenium preparation. [Diagram 2] 1 is a graph showing changes in serum glutathione peroxidase activity following multiple administrations of a selenium preparation. [Diagram 3] FIG. 1 is a comparative diagram of the toxicity of each selenium product to rat liver. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The technical solutions of the present invention will be described clearly and completely below with reference to the examples of the present invention. It should be noted that the specific embodiments described in this specification are only used to describe and interpret the present invention, and are not used to limit the present invention.

[0019] In the following examples, all methods are conventional unless otherwise specified, and all of the reagents and materials are commercially available unless otherwise specified.

[0020] Example 1 The process for producing selenoprotein derived from Cardamine violifolia in this example is specifically as follows. (1) Cardamine violifolia was dried and crushed, then sieved through a 40-mesh sieve. Cardamine violifolia powder and cellulase (mass ratio of the two was 1:0.02) were added to pure water, and the mixture was shaken at 40°C for 2 hours to effect enzymatic hydrolysis. The mixture was then heated to 85°C to inactivate the enzyme, and centrifuged at 3000 r / min for 15 minutes to remove precipitated impurities and obtain the supernatant. (2) A complex enzyme consisting of alkaline protease, trypsin, papain, protease K, and protease XIV (1:1:1:1:1) was added to the supernatant so that the mass ratio of the complex enzyme to the Cardamine violifolia powder was 0.015:1. The supernatant to which the complex enzyme was added was placed in an electrodialysis device with the following operating parameters: voltage 15V, current 3.3A, electrode water flow rate 12L / h, fresh water and concentrated water flow rate 20L / h, fresh water to concentrated water volume ratio 1:1, and 2% sodium sulfate solution as electrode water, and electrodialysis treatment was performed simultaneously. In this process, the temperature of the enzyme hydrolysis reaction solution was maintained at 45°C and the reaction was performed for 2h. After the reaction was completed, the temperature was raised to 85°C to inactivate the enzyme, and the filtrate was collected. (3) The filtrate was concentrated in vacuo and then spray-dried to give the desired product.

[0021] Example 2 The process for producing selenoprotein derived from Cardamine violifolia in this example is specifically as follows. (1) Cardamine violifolia was dried and crushed, then sieved through a 40-mesh sieve. Cardamine violifolia powder and cellulase (mass ratio of the two was 1:0.02) were added to pure water, and the mixture was shaken at 40°C for 2 hours to effect enzymatic hydrolysis. The mixture was then heated to 85°C to inactivate the enzyme, and centrifuged at 3000 r / min for 15 minutes to remove precipitated impurities and obtain the supernatant. (2) A complex enzyme consisting of alkaline protease, trypsin, papain, protease K, and protease XIV (1:1:1:1:1) was added to the supernatant. Here, the mass ratio of the amount of the complex enzyme to the Cardamine violifolia powder was 0.015:1. The supernatant to which the complex enzyme was added was placed in an electrodialysis device with the following operating parameters: voltage 13V, current 3A, electrode water flow rate 15L / h, fresh water and concentrated water flow rate 22L / h, fresh water to concentrated water volume ratio 1:1, and 2% sodium sulfate solution as electrode water, and electrodialysis treatment was performed simultaneously. In this process, the temperature of the enzyme hydrolysis reaction solution was maintained at 45°C and the reaction was performed for 2h. After the reaction was completed, the temperature was raised to 85°C to inactivate the enzyme, and the filtrate was collected. (3) The filtrate was concentrated in vacuo and then spray-dried to give the desired product.

[0022] Comparative Example 1 The process for producing selenoprotein derived from Cardamine violifolia in this example is specifically as follows. (1) Cardamine violifolia was dried and crushed, then sieved through a 40-mesh sieve. Cardamine violifolia powder and cellulase (mass ratio of the two was 1:0.02) were added to pure water, and the mixture was shaken at 40°C for 2 hours to effect enzymatic hydrolysis. The mixture was then heated to 85°C to inactivate the enzyme, and centrifuged at 3000 r / min for 15 minutes to remove precipitated impurities and obtain the supernatant. (2) A complex enzyme consisting of alkaline protease, trypsin, papain, protease K, and protease XIV (1:1:1:1:1) was added to the supernatant. The mass ratio of the complex enzyme to the Cardamine violifolia powder was 0.015:1, and the temperature of the enzymatic hydrolysis reaction solution was maintained at 45°C and the reaction was allowed to proceed for 2 hours. After the reaction was completed, the temperature was raised to 85°C to inactivate the enzymes, and the filtrate was collected. (3) The filtrate was concentrated in vacuo and then spray-dried to give the desired product.

[0023] Comparative Example 2 The process for producing selenoprotein derived from Cardamine violifolia in this example is specifically as follows. (1) Cardamine violifolia was dried and crushed, then sieved through a 40-mesh sieve. Cardamine violifolia powder and cellulase (mass ratio of the two was 1:0.02) were added to pure water, and the mixture was shaken at 40°C for 2 hours to effect enzymatic hydrolysis. The mixture was then heated to 85°C to inactivate the enzyme, and centrifuged at 3000 r / min for 15 minutes to remove precipitated impurities and obtain the supernatant. (2) The supernatant was placed in an electrodialysis device with operating parameters of 15 V voltage, 3.3 A current, 12 L / h electrode water flow rate, 20 L / h fresh water and concentrated water flow rates, 1:1 volume ratio of fresh water to concentrated water, and 2% sodium sulfate solution as electrode water, and electrodialysis treatment was performed. In this process, the temperature of the reaction solution was maintained at 45°C and the reaction was allowed to proceed for 2 hours. After the reaction was completed, the filtrate was collected. (3) The filtrate was concentrated in vacuo and then spray-dried to give the desired product.

[0024] Comparative Example 3 The process for producing selenoprotein derived from Cardamine violifolia in this example is specifically as follows. (1) Cardamine violifolia was dried and crushed, then sieved through a 40 mesh sieve. The Cardamine violifolia powder was added to pure water and extracted by shaking at 40°C for 2 hours. The extract was then centrifuged at 3000 r / min for 15 minutes to remove precipitated impurities and obtain the supernatant. (2) The supernatant was placed in an electrodialysis device with operating parameters of 15 V voltage, 3.3 A current, 12 L / h electrode water flow rate, 20 L / h fresh water and concentrated water flow rates, 1:1 volume ratio of fresh water to concentrated water, and 2% sodium sulfate solution as electrode water, and electrodialysis treatment was performed. In this process, the temperature of the reaction solution was maintained at 45°C and the reaction was allowed to proceed for 2 hours. After the reaction was completed, the filtrate was collected. (3) The filtrate was concentrated in vacuo and then spray-dried to give the desired product.

[0025] Comparative Example 4 The process for producing selenoprotein derived from Cardamine violifolia in this example is specifically as follows. (1) Cardamine violifolia was dried and crushed, then sieved through a 40-mesh sieve. Cardamine violifolia powder and cellulase (mass ratio of the two was 1:0.02) were added to pure water, and the mixture was shaken at 40°C for 2 hours to effect enzymatic hydrolysis. The mixture was then heated to 85°C to inactivate the enzyme, and centrifuged at 3000 r / min for 15 minutes to remove precipitated impurities and obtain the supernatant. (2) A complex enzyme consisting of alkaline protease, trypsin, papain, protease K, and protease XIV (1:1:1:1:1) was added to the supernatant. The mass ratio of the complex enzyme to the Cardamine violifolia powder was 0.015:1, and the temperature of the enzymatic hydrolysis reaction solution was maintained at 45°C and the reaction was allowed to proceed for 2 hours. After the reaction was completed, the temperature was raised to 85°C to inactivate the enzymes, and the filtrate was collected. (3) The filtrate from the previous step was treated by electrodialysis, and the operating parameters of the electrodialysis apparatus were: voltage 15V, current 3.3A, electrode water flow rate 12L / h, fresh water and concentrated water flow rate 20L / h, fresh water to concentrated water volume ratio 1:1, and 2% sodium sulfate solution as electrode water. In this process, the temperature of the reaction solution was maintained at 45°C and the reaction was carried out for 2 hours. After the reaction was completed, the filtrate was collected. (4) The filtrate was concentrated in vacuum and then spray-dried to give the desired product.

[0026] Comparative Example 5 The process for producing selenoprotein derived from Cardamine violifolia in this example is specifically as follows. (1) Cardamine violifolia was dried and crushed, then sieved through a 40-mesh sieve. Cardamine violifolia powder and cellulase (mass ratio of the two was 1:0.02) were added to pure water, and the mixture was shaken at 40°C for 2 hours to effect enzymatic hydrolysis. The mixture was then heated to 85°C to inactivate the enzyme, and centrifuged at 3000 r / min for 15 minutes to remove precipitated impurities and obtain the supernatant. (2) A complex enzyme consisting of alkaline protease, trypsin, protease E, neutral protease and protease K (1:1:1:1:1) was added to the supernatant. Here, the mass ratio of the amount of the complex enzyme to the Cardamine violifolia powder was 0.015:1. The supernatant to which the complex enzyme was added was placed in an electrodialysis device with the following operating parameters: voltage 15V, current 3.3A, electrode water flow rate 12L / h, fresh water and concentrated water flow rate 20L / h, fresh water to concentrated water volume ratio 1:1, and 2% sodium sulfate solution as electrode water, and electrodialysis treatment was performed simultaneously. In this process, the temperature of the enzyme hydrolysis reaction solution was maintained at 45°C and the reaction was performed for 2h. After the reaction was completed, the temperature was raised to 85°C to inactivate the enzymes, and the filtrate was collected. (3) The filtrate was concentrated in vacuo and then spray-dried to give the desired product.

[0027] The selenoproteins derived from Cardamine violifolia produced in Examples 1-2 and Comparative Examples 1-5 were subjected to detection including the following two parts.

[0028] (1)Component analysis The protein content in each selenoprotein sample was determined by the method of GB5009.5, the ash content by the method of GB5009.4, the total selenium content by the method of GB5009.93, the organic selenium content by the method of DSB42 / 002, the ratio of selenocysteine ​​(converted into selenocystine; free selenocysteine ​​is extremely unstable, so two molecules of selenocysteine ​​combine to form one molecule of selenocystine) by the method of T / CHC1001, the lead content by the method of GB5009.75, the arsenic content by the method of GB5009.76, the cadmium content by the method of GB5009.12, and the mercury content by the method of GB5009.17. The results are shown in the following table. JPEG0007608004000002.jpg89170

[0029] From the data of the examples and comparative examples, it can be seen that electrodialysis can effectively reduce the content of various heavy metals, inorganic selenium and ash, thereby increasing the content of protein in the sample, and the simultaneous use of complex enzyme decomposition and electrodialysis treatment can significantly increase the content of selenocystine. The reasons for this are considered to be as follows. First, the combination of enzyme decomposition can be used to more fully enzymatically decompose the components of Cardamine violifolia, which can fully dissociate the heavy metals complexed with proteins; during the process of enzymatic decomposition, the precipitation of metal ions occurs gradually, so that electrodialysis can be performed for a sufficient time to fully remove the heavy metal components; and the simultaneous process of enzyme decomposition + electrodialysis can quickly remove the dissociated heavy metal components, thereby reducing the structural changes of bioactive components such as selenocystine in the heavy metal environment, and maximally maintaining the stability and bioactivity of selenocystine. Next, the combination of enzymatic degradation designed in the present application can more fully expose or separate the protein derived from Cardamine violifolia from the tissue of Cardamine violifolia plant, and the combination of enzymatic degradation can more fully enzymatically decompose the protein derived from Cardamine violifolia into various peptide or amino acid fragments, thereby maximally releasing selenocystine and maintaining the most stable state in this enzymatic degradation system. In addition, since the active center of human selenoprotein is selenocysteine, maintaining the activity of selenoprotein / selenoenzyme with selenocysteine ​​as the active center is important for adjusting the antioxidant balance in humans, but the presence of heavy metals often changes the quaternary structure of protease and reduces protein activity, so the above method can further reduce the influence of heavy metals on the activity of selenoprotein characterized by selenocysteine / selenocystine, and further improve physiological activity while ensuring safety.

[0030] The selenoprotein standard sample prepared by the present invention has a high total selenium content of 1500-5000mg / kg, a high organic selenium content of more than 99%, a selenocystine content of more than 90%, a high protein content of more than 60%, an ash content of less than 2%, and undetectable heavy metals such as lead, arsenic, mercury, and cadmium. The above indicators (except for the organic selenium form being selenocystine) and other indicators are in full compliance with the requirements of GB1903.28-2018 National Food Safety Standard for Food Nutrition Enhancer Selenoprotein.

[0031] (2) Animal experiments on antioxidant activity In order to verify the enzyme activity of the standard samples of Cardamine violifolia-derived selenoproteins prepared in the present invention, specifically, the Cardamine violifolia-derived selenoproteins prepared in Example 1 and Comparative Example 1 were used to conduct an antioxidant animal test according to the following protocol. The test consisted of four test sample dose groups and one control group. The four dose groups were the low dose group of Comparative Example 1, the high dose group of Comparative Example 1, the low dose group of Example 1, and the high dose group of Example 1, and the doses (in selenium equivalent) were 0.03 mgSe / kg, 0.3 mgSe / kg, 0.02 mgSe / kg, and 0.2 mgSe / kg, respectively.

[0032] The test substance was prepared into a solution, mixed with the feed, and orally administered, with all animals completing oral intake within 1 minute. The control group was given an equal volume of distilled water. The number of animals in each test substance group was 4, and the control group was 2. The administration of the test substance was divided into two administration cycles, with one administration cycle being 7 consecutive days, and the administration frequency was once a day. A dissolution period was provided between the two administration periods, which was 8 days for the Comparative Example 1 group and 10 days for the Example 1 group.

[0033] To observe the drug exposure level in the animals, about 5mL of whole blood was collected from the anterior vena cava and added to a blood collection tube labeled with K2EDTA as an anticoagulant. The detection indicator was serum glutathione peroxidase (GPX) activity after single and multiple administration of selenium preparations (liver homogenates were prepared according to the kit guidelines and detected using a kit from Nanjing Jiancheng Biological Co., Ltd.).

[0034] The detection results are shown in Figures 1 and 2. Specifically, when the selenoprotein derived from Cardamine violifolia prepared in Comparative Example 1 was administered once or multiple times, there was no significant effect on the plasma GPX activity, when 0.14 mg Se / kg of the selenoprotein derived from Cardamine violifolia prepared in Example 1 was administered once, the plasma GPX activity could be significantly increased, and when 0.03 mg Se / kg and 0.14 mg Se / kg of the selenoprotein derived from Cardamine violifolia prepared in Example 1 were administered multiple times, the plasma GPX activity could be significantly increased. Thus, the selenoprotein derived from Cardamine violifolia prepared in Example 1 could significantly improve the plasma GPX activity, and the selenoprotein derived from Cardamine violifolia prepared in Comparative Example 1 had no significant effect on the plasma GPX activity.

[0035] (3) Safety animal testing In this study, male rats were repeatedly orally administered the selenoprotein derived from Cardamine biolifolia produced in the present invention (specifically, the one produced in Example 1), sodium selenite, selenium-rich yeast, and the selenoprotein sample produced in Comparative Example 3 to observe and investigate the potential toxicity. (The daily safety limit of selenium for adults is 400μg / person, and the safe dose in this study is equivalent to 96μgSe / kg BW for humans, which is equivalent to 5760μg / day of selenium for an adult body weight of 60kg, which is nearly 15 times the daily safety limit for adults.)

[0036] After 3 months of administration, the liver lesions of rats in each experimental group are shown in Figure 3. A is the blank group, B is the sodium selenite group, C is the selenium-rich yeast group, D is the selenoprotein group prepared in Comparative Example 3, and E is the Cardamine biolifolia-derived selenoprotein group prepared in Example 1. As can be seen, the sodium selenite group caused liver lesions, and the selenium-rich yeast group and the selenoprotein prepared in Comparative Example 3 also showed lesions, although to different degrees, while the Cardamine biolifolia-derived selenoprotein group prepared in the present invention was consistent with the blank control group and showed no toxic effect on the liver, proving that the Cardamine biolifolia-derived selenoprotein prepared in the present invention is not toxic at the above doses.

[0037] The selenoprotein sample prepared in Comparative Example 3 also caused some lesions, the reason for which was related to the preparation method, in which the obtained selenoprotein sample contained certain heavy metals and had too high ash content, resulting in liver damage.

[0038] The selenium form in selenium-rich yeast is mainly selenomethionine, which causes various degrees of liver lesions, but approximately 80% of the selenium form in the selenoprotein produced by this invention is selenocysteine / selenocystine, which has no toxic effect on the liver. This further proves that the safety of selenoprotein derived from Cardamine violifolia, which contains selenocysteine / selenocystine as its characteristic components, is higher than that of selenium-rich yeast, which contains selenomethionine as its main component, and is also much higher than that of inorganic selenium (sodium selenite).

[0039] Furthermore, rats were given different doses of the standard sample of selenoprotein derived from Cardamine violifolia (prepared in Example 1). The doses were 0.15, 0.30, and 0.60 mgSe / kg, calculated as selenium content, respectively. The total sperm count and motility rate of each dose group are shown in the following table. JPEG0007608004000003.jpg42170

[0040] As can be seen from the above table, no significant changes were observed in the total sperm count and motility rate at each dose, proving that the Cardamine biolifolia-derived selenoprotein produced in this invention has high safety.

[0041] In conclusion, the Cardamine biolifolia-derived selenoprotein standard sample produced in this invention has technical indicators that fully meet or significantly exceed the requirements of the national standard for selenoproteins, can significantly improve the biological activity of glutathione peroxidase (GPX) in plasma, and can further improve the antioxidant capacity. It is also safe and is expected to fill the gap in the market for selenoprotein standard products.

[0042] The above is a preferred embodiment of the present invention, and does not limit the scope of the rights of the present invention. Furthermore, any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention by those skilled in the art should be included in the scope of protection of the present invention.

Claims

1. A method for producing a highly active and safe standard sample of selenoprotein derived from Cardamine biolifolia, comprising the steps of: Step 1, hydrolyzing Cardamine biolifolia powder with cellulase, inactivating the enzyme after completion of enzymatic hydrolysis, and centrifuging to obtain a supernatant; Step 2: adding a complex enzyme consisting of alkaline protease, trypsin, papain, protease K, and protease XIV to the supernatant to continue the enzymatic decomposition treatment, and simultaneously carrying out electrodialysis to dissociate heavy metals complexed with proteins, and then removing heavy metal ions and inorganic selenium salt ions by electrodialysis, followed by inactivating the enzymes; Step 3 is to concentrate the reaction solution obtained in step 2 and then dry it to obtain a selenoprotein standard sample; A method for producing a selenoprotein standard sample, comprising:

2. 2. The method for producing a selenoprotein standard sample according to claim 1, wherein in step 1, the amount of cellulase added is 0.1-10 wt % of the Cardamine violifolia powder, and the enzymatic decomposition by cellulase is performed at a temperature of 30-50° C. for 1-4 hours.

3. 2. The method for producing a selenoprotein standard sample according to claim 1, wherein in step 1, the centrifugation parameters are 1,000-10,000 r / min and 5-60 min.

4. 2. The method for producing a selenoprotein standard sample according to claim 1, wherein in step 2, the amount of the conjugated enzymes added is 0.1-10 wt % of the Cardamine violifolia powder, and the enzymatic decomposition by the conjugated enzymes is performed at a temperature of 30-50° C.

5. 2. The method for preparing a selenoprotein standard sample according to claim 1, wherein in step 2, the parameters of the electrodialysis are: voltage 15-100 V, current 10 A or less, electrode water flow rate 1-100 L / h, fresh water and concentrated water flow rates 1-100 L / h, fresh water and concentrated water volume ratio 1:1, and the electrode water is a 0.1-10 wt % sodium sulfate solution.

6. The method for producing a selenoprotein standard sample according to claim 5, wherein the electrodialysis treatment time is 0.5 to 8 hours.

7. 2. The method for producing a selenoprotein standard sample according to claim 1, wherein in step 2, the temperature of the enzyme inactivation treatment is 85 to 95° C.

8. 2. The method for producing a selenoprotein standard sample according to claim 1, wherein in step 3, the drying method is spray drying or freeze drying.

9. 9. Use of a highly active and safe Cardamine biolifolia-derived selenoprotein standard sample obtained by the method according to any one of claims 1 to 8 in the production of food or pharmaceutical products.

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