Celenium chelate pea oligopeptide and its preparation method and uses

Abstract

Selenium Chelated Pea Oligopeptide and its preparation method and use. Selenium Chelated Pea Oligopeptide After at least one of the following three digestion methods, the selenium content does not exceed 3% of the change rate before digestion: hydrolysis with pepsin at pH 2 and temperature 37°C for 4 hours; hydrolysis with trypsin at pH 7.5 and temperature 37°C for 6 hours; or hydrolysis with pepsin at pH 2 for 4 hours, and then hydrolysis with trypsin at pH 6.8 for 6 hours, all at a constant temperature of 37°C. Selenium Chelated Pea Oligopeptide The method involves reacting an aqueous solution of the compound with sodium selenite, followed by alcohol precipitation and drying.

Description

[Technical Field]

[0001] The present invention Selenium Chelate Pea Oligopeptide Regarding its preparation method and uses, in particular, it is highly stable, effectively absorbed by humans, and has excellent antioxidant properties. Selenium Chelate Pea Oligopeptide And relating to its manufacturing techniques and uses. [Background technology]

[0002] According to traditional metabolic models, proteins are hydrolyzed into amino acids before being absorbed and utilized by living organisms. However, recent research has shown that animals' protein needs also depend on a certain amount of small molecule active peptides, and that oligopeptides have dedicated carriers and absorption pathways in the human gut, allowing them to enter the small intestine in their complete form and be absorbed.

[0003] Pea oligopeptides are small molecule peptides obtained after enzymatic degradation or hydrolysis of pea proteins. It has already been reported that pea oligopeptides are physiologically active peptides with a wide range of applications, possessing antibacterial, antioxidant, and ACE inhibitory activities.

[0004] Selenium is one of the trace elements that humans must take in from the outside world, and it plays an important role in maintaining human life activities. Research has shown that selenium deficiency can weaken the human immune system and may cause diseases such as Keshan disease.

[0005] Currently, in addition to normal food intake, a common method of selenium supplementation is the oral administration of inorganic selenium such as sodium selenite. However, research has shown that organic selenium is less toxic and has a higher absorption rate than inorganic selenium. As a result, there are numerous reports on research into related oligopeptide selenium-based organic selenium products such as soy peptide selenium chelate, reishi mushroom peptide selenium chelate, and fish head protein peptide selenium chelate. However, there are few reports on pea oligopeptides or selenium chelation. Meanwhile, the molecular stability of the resulting oligopeptide selenium (usually called chelated selenium) obtained by introducing selenium into oligopeptide molecules is considered a key issue in this type of product research. The question is whether to use oligopeptide selenium directly as a product to ensure effective selenium supplementation, or to use oligopeptide selenium as an intermediate raw material for further processing to guarantee the expected effects of subsequent processing. However, the goal is to create a product that is effectively absorbed by humans and can be stably combined. Selenium Chelate Pea Oligopeptide That is even rarer. [Overview of the project] [Problems that the invention aims to solve]

[0006] In contrast to the aforementioned shortcomings of conventional technology, the present invention provides stable performance and effective absorption by humans through the intestines, thereby achieving effective selenium supplementation. Selenium Chelate Pea Oligopeptide To provide.

[0007] The present invention Selenium Chelate Pea Oligopeptide The present invention provides a method for preparing the above, and by adopting this preparation method, Selenium Chelate Pea Oligopeptide In addition to obtaining the necessary compounds, it also has the advantage of a high chelation rate and high yield.

[0008] The present invention also relates to the above Selenium Chelate Pea Oligopeptide It also offers applications in healthcare foods. [Means for solving the problem]

[0009] To achieve the above objectives, the present invention provides Selenium Chelate Pea Oligopeptide teeth, A method using pepsin and performing hydrolysis for 4 hours under conditions of a pH value of 2 and a temperature of 37°C. A method using trypsin and hydrolysis performed for 6 hours under conditions of a pH of 7.5 and a temperature of 37°C. After at least one of three digestion methods—one in which the temperature is kept constant at 37°C, hydrolysis is performed for 4 hours using pepsin under conditions where the pH value is 2, and the other in which hydrolysis is performed for another 6 hours using trypsin under conditions where the pH value is 6.8—the selenium content does not exceed 3% of the change rate before the digestion treatment.

[0010] Provided by the present invention Selenium Chelate Pea Oligopeptide (Also known as pea oligopeptide selenium chelate) shows that after the above digestion treatment, the selenium content does not exceed 3% compared to the rate of change (more precisely, the rate of decrease) before digestion treatment. Furthermore, studies have shown that almost the vast majority of selenium is combined with pea oligopeptides via covalent bonds (more precisely, coordinate bonds), and that the covalent bonds between the two are relatively stable. This demonstrates that the selenium element passes stably through the stomach and intestines along with the pea oligopeptides and is ultimately absorbed indirectly through complete absorption by the small intestinal mucosa into the pea oligopeptides.

[0011] Furthermore, Selenium Chelate Pea Oligopeptide It exhibits excellent thermal stability; after heat treatment at 100°C or below for 2 hours, no significant difference in selenium content compared to before heat treatment is observed, and the rate of change does not exceed 3%. That is, Selenium Chelate Pea Oligopeptide A system in which the substance is dispersed in water is treated at a temperature below 100°C for 2 hours, after which the selenium content is 97% or more of that of the control group. This can further ensure the digestion and absorption of selenium in humans, and when used as an intermediate raw material for further processing, it can also guarantee the expected effects of subsequent processing.

[0012] Furthermore, Selenium Chelate Pea Oligopeptide It exhibits excellent acid-base stability, and after 2 hours of treatment under conditions of pH 3-11 and temperature 37°C, the selenium content does not change by more than 25% compared to the control ratio before treatment. That is, Selenium Chelate Pea Oligopeptide The system in which it is dispersed in water has a selenium content of 75% or more of that of the control group without acid-base treatment after being treated under acidic, alkaline or neutral conditions for 2 h. Due to its excellent acid-base stability, Selenium Chelate Pea Oligopeptide If it can further ensure the digestion and absorption of selenium in the human body in

[0013] At the same time, the Selenium Chelate Pea Oligopeptide has a much improved ability to remove DPPH free radicals and hydroxyl free radicals (·OH) compared with the raw material pea oligopeptide, and the Selenium Chelate Pea Oligopeptide also has relatively strong antioxidant and reducing abilities, and its reducing ability is not a simple superposition of pea oligopeptide and sodium selenite, and thus, Selenium Chelate Pea Oligopeptide is very suitable for being developed as antioxidant health food and selenium-supplemented health food.

[0014] In some embodiments of the present invention, the provided Selenium Chelate Pea Oligopeptide has an acid-soluble protein content higher than 23% and a total nitrogen content higher than 23%, which indicates that Selenium Chelate Pea Oligopeptide the molecular weight of the protein in

[0015] Specifically, in some embodiments of the present invention, Selenium Chelate Pea Oligopeptide in

[0016] Furthermore, Selenium Chelate Pea Oligopeptide contains PPKIYP (Pro-Pro-Lys-Ile-Tyr-Pro), and by identification, PPKIYP is a peptide fragment with relatively strong antioxidant ability.

[0017] After quantitative analysis, Selenium Chelate Pea OligopeptideThis includes a peptide fragment PPKIYP with a mass content of 25 ng / mg or more, and is generally 25-35 ng / mg.

[0018] moreover, Selenium Chelate Pea Oligopeptide It also includes the peptide fragments TGRGAP (Thr-Gly-Arg-Gly-Ala-Pro), HQMPKP (His-Gln-Met-Pro-Lys-Pro), and TSSLP (Thr-Ser-Ser-Leu-Pro).

[0019] After quantitative analysis, Selenium Chelate Pea Oligopeptide In this product, the mass content of TGRGAP is typically 25 ng / mg or more, generally between 25 and 35 ng / mg; the mass content of HQMPKP is typically 50 ng / mg or less, generally between 40 and 50 ng / mg; and the mass content of TSSLP is generally between 1 and 5 ng / mg.

[0020] Furthermore, in some embodiments of the present invention, the selenium content in the pea oligopeptide is 0.08 g / 100 g or more, that is, Selenium Chelate Pea Oligopeptide The mass of selenium is 0.08g or more per 100g, which further ensures the selenium supplementation effect.

[0021] In some embodiments of the present invention, Selenium Chelate Pea Oligopeptide The product may be the result of a reaction between an aqueous solution of pea oligopeptide and sodium selenite (Na2SeO3). Specifically, an aqueous solution of pea oligopeptide and sodium selenite are mixed, and the reaction is carried out at 60-90°C for 20 minutes or more. The resulting reaction product is then subjected to alcohol precipitation and drying. Selenium Chelate Pea Oligopeptide You will come to obtain it.

[0022] As the pea oligopeptide described above, it is optimal to select from pea oligopeptides with a molecular weight less than 1000u and a component content of more than 80%, and in particular, it may be selected from pea oligopeptides with a molecular weight less than 1000u and a component content of 90% or more. In some examples of the present invention, the pea oligopeptide can be obtained by the following preparation method: Mix pea protein powder (with a protein content of more than 80%) with water so that the ratio of material to liquid is 1:8 to 12, adjust the pH value of the material liquid to 8 to 10, and control the temperature to 40 to 60°C. For enzymatic decomposition Alkaline protease and neutral protease are added, with the dosage of both enzymes being 1.0-3.0% of the pea protein powder mass, and the enzymatic decomposition time being 3-6 hours. After the enzymatic decomposition is complete, the enzymes in the liquid material are inactivated, centrifuged, filtered through a ceramic membrane (with a pore size of 50-200 nm), vacuum concentrated, sterilized, and finally dried by spraying to form a powder, thereby preparing pea oligopeptide powder.

[0023] The present invention further, Selenium Chelate Pea Oligopeptide The present invention provides a method for preparing a product, in which an aqueous solution of pea oligopeptide is mixed with sodium selenite, the resulting mixed system is reacted at 60-90°C for 20 minutes or more, and the resulting reaction product is subjected to alcohol precipitation and drying. Selenium Chelate Pea Oligopeptide This includes the step of obtaining [something].

[0024] By detecting the reaction between pea oligopeptide and sodium selenite using methods such as full-wavelength ultraviolet scanning, scanning electron microscopy, and Fourier infrared spectroscopy, a new chelate was obtained. It is presumed that selenium in sodium selenite and pea oligopeptide are stably bonded together via covalent bonds. 4+ )The carboxyl group and amino group in the pea oligopeptide may be bonded in a coordinate bond manner. That is, Se 4+This provides a 4d empty orbital, and O and N provide lone pairs of electrons, each occupying the 4d empty orbital to form a stable coordination bond, thereby, Selenium Chelate Pea Oligopeptide It has excellent thermal stability, acid-base stability, and in vitro digestion stability, ensuring that selenium can enter the intestines smoothly and be indirectly absorbed by ingesting oligopeptides in the intestinal mucosa, thereby achieving a selenium supplementation effect. Selenium Chelate Pea Oligopeptide When used as an intermediate raw material for further processing, it also becomes possible to guarantee the expected effects of subsequent processing.

[0025] For the reasons stated above, 、 The above reaction may also be called a "chelation reaction," so the obtained Selenium Chelate Pea Oligopeptide However, following the usual terminology in this field, it may also be called "pea oligopeptide selenium chelate" or "pea oligopeptide chelated selenium," where the combination ratio of selenium and pea oligopeptide is also indicated by the "chelation ratio."

[0026] Temperature significantly affects the reaction between pea oligopeptide and sodium selenite; as the temperature increases, the chelation rate and Selenium Chelate Pea Oligopeptide The yields in all cases tend to increase before decreasing again. In the specific implementation of the present invention, the reaction temperature is generally controlled to 70-90°C, and more specifically to 80-85°C, in order to obtain a higher chelation rate and yield simultaneously.

[0027] In some embodiments of the present invention, the mass ratio of pea oligopeptide to sodium selenite (mass ratio of peptide salts) is generally controlled to be 1 to 5:1. During the chemical reaction process, the relative ratio of the reactants can affect the formation of several space keys. Specifically, in some embodiments of the present invention, as the ratio of pea oligopeptide to sodium selenite increases (e.g., from 1:1 to 5:1), the chelation rate gradually decreases, Selenium Chelate Pea OligopeptideThe yield increases before decreasing again. Considering the chelation rate and yield together, the mass ratio of pea oligopeptide to sodium selenite is generally controlled to be between 2 and 4:1.

[0028] In some embodiments of the present invention, the concentration of pea oligopeptide (peptide concentration) in the aqueous solution of pea oligopeptide is generally controlled to be 1-5 g / 100 mL. As the peptide concentration increases, the chelation rate and Selenium Chelate Pea Oligopeptide In all cases, the levels increase, then stabilize, and then show a decreasing trend. Considering the chelation rate and yield as a whole, the peptide concentration is generally controlled to be 3-5 g / 100 mL.

[0029] Rationally controlling the pH of the mixed system of pea oligopeptide and sodium selenite is advantageous for obtaining higher chelation rates and yields. In some examples of the present invention, it is common to carry out the chelation reaction under weakly alkaline conditions, such as controlling the pH to 7.5-9. When the pea oligopeptide and sodium selenite are completely dissolved, the pH of the mixed system is about 8, so it is not necessary to adjust the pH, or the pH may be adjusted by adding NaOH or acetic acid before the reaction.

[0030] Rationally controlling the reaction time is also advantageous in obtaining the highest possible chelation rate and yield. Generally, as the reaction time is extended, both the chelation rate and yield tend to increase, then decrease again, and finally gradually stabilize. In the specific implementation of this invention, considering the chelation rate, yield, and time cost as a whole, the reaction time is generally controlled within 20 min to 60 min.

[0031] As understood, the pea oligopeptide used as a reaction raw material also has a molecular weight distribution. Selenium Chelate Pea OligopeptideThis affects the final molecular weight distribution, and in the specific implementation of the present invention, the content of the selected pea oligopeptide with a molecular weight less than 1000u is optimally higher than 80%, and preferably 90% or more.

[0032] In some embodiments of the present invention, the pea oligopeptide described above is obtained by employing the following method: mixing pea protein powder (with a protein content of more than 80%) with water in a material-to-liquid ratio of 1:8 to 12, adjusting the pH of the material liquid to 8 to 10, and controlling the temperature to 40°C to 60°C. For enzymatic decomposition This method involves adding alkaline protease and neutral protease, with the dosage of both proteases being 1.0-3.0% of the pea protein powder mass, and the enzymatic decomposition time being 3-6 hours. After the enzymatic decomposition is complete, the enzymes in the liquid material are inactivated, followed by centrifugation, filtration through a ceramic membrane (ceramic membrane pore size 50-200 nm), vacuum concentration, sterilization, and drying by spraying to produce a powder. This process allows for the production of pea oligopeptide powder that satisfies the above requirements.

[0033] The invention further states the above Selenium Chelate Pea Oligopeptide It provides applications in healthcare foods. Selenium Chelate Pea Oligopeptide It has excellent stability, can enter the intestines smoothly, and is indirectly absorbed by the intestinal mucosa through the intake of oligopeptides, thereby achieving the selenium supplementation effect. Selenium Chelate Pea Oligopeptide It may be developed as a novel selenium supplementation preparation for use in nutritional and functional foods for people with selenium deficiency. For example, it may be added to milk powder or other healthcare foods, or further processed as an intermediate ingredient to obtain healthcare foods. Selenium Chelate Pea Oligopeptide Its ability to remove DPPH free radicals and hydroxyl free radicals is significantly improved compared to the raw material, pea oligopeptide, and it possesses relatively strong reducing and antioxidant functions, making it particularly suitable for the development of antioxidant healthcare foods and antioxidant selenium supplements.

[0034] According to the present invention Selenium Chelate Pea Oligopeptide Because selenium is covalently bonded to pea oligopeptides and possesses relatively strong bonding forces, it exhibits excellent stability depending on the digestion method. After hydrolysis with pepsin and trypsin, the decrease in selenium content is small, allowing selenium to enter the intestines smoothly and be indirectly absorbed by the intestinal mucosa through the intake of oligopeptides. This achieves a selenium supplementation effect, making it suitable for application in selenium supplementation healthcare foods and for processing as an intermediate raw material to obtain desired products.

[0035] Furthermore, Selenium Chelate Pea Oligopeptide Furthermore, the proportion of components with a molecular weight less than 1000u is higher than 85%, and the selenium content is 0.08g / 100g or more, which makes it possible to further improve the selenium supplementation effect.

[0036] Furthermore, Selenium Chelate Pea Oligopeptide The ability to remove DPPH free radicals and OH free radicals is significantly improved compared to the raw material, pea oligopeptide. Selenium Chelate Pea Oligopeptide Furthermore, because it has relatively strong antioxidant and reducing capabilities, Selenium Chelate Pea Oligopeptide This can be applied to antioxidant healthcare foods. [Effects of the Invention]

[0037] According to the present invention Selenium Chelate Pea Oligopeptide The preparation method obtained Selenium Chelate Pea Oligopeptide In this case, selenium and oligopeptide are linked by a stable covalent bond. Selenium Chelate Pea Oligopeptide This ensures excellent thermal stability, acid-base stability, and digestion stability, as well as relatively strong antioxidant and reducing capabilities. Furthermore, the reducing capability is not simply due to the superposition of the chelated raw material peptide and sodium selenite, but is enhanced by the preparation method.

[0038] at the same time, Selenium Chelate Pea OligopeptideBy adopting this preparation method, the following advantages are obtained: Because the moisture content is relatively low, generally 14.17% ± 1.12%, most molds and Staphylococcus bacteria are inhibited, making storage easier. The acid-soluble protein content is 23% or more, and the total nitrogen content is 23% or more, which means... Selenium Chelate Pea Oligopeptide This indicates that the molecular weight of the proteins in this sample is low. More than 85% of the molecular weight distribution is below 1000u, and most of these are below 500u, making it very favorable for absorption by humans. The selenium content is 0.08g / 100g or more, ensuring the selenium supplementation effect. [Brief explanation of the drawing]

[0039] [Figure 1] This is a diagram of the selenium standard curve drawn to measure the selenium content of the present invention. [Figure 2] This is a gel chromatogram showing the molecular weight distribution of the selen chelate pea oligopeptide in Example 1 of the present invention. [Figure 3] This is a full-wavelength ultraviolet scan diagram of pea oligopeptide and selenium chelate pea oligopeptide in Example 1 of the present invention. [Figure 4] This is a scanning electron microscope image (×1000) of the pea oligopeptide and selenium chelate pea oligopeptide in Example 1 of the present invention. [Figure 5] This is an infrared spectral diagram of pea oligopeptide and selenchelate pea oligopeptide in Example 1 of the present invention. [Figure 6] This figure shows the selenium content of selenium chelate pea oligopeptide treated under different temperature conditions in Example 1 of the present invention. [Figure 7] This figure shows the selenium content of selenium chelate pea oligopeptide treated under different acid-base conditions in Example 1 of the present invention. [Figure 8]This is the selenium content of the selenium chelate pea oligopeptide treated with different digestion methods in Example 1 of the present invention. [Figure 9] This is a comparative diagram of DPPH free radical removal using selen chelate pea oligopeptide and its synthesis raw materials in Example 1 of the present invention. [Figure 10] This is a comparison diagram of DPPH free radical removal by VC. [Figure 11] This is a comparative diagram of OH free radical removal using selen chelate pea oligopeptide and its synthesis raw materials in Example 1 of the present invention. [Figure 12] This is a comparison diagram of OH free radical removal by VC. [Figure 13] This is a comparative diagram of the reducing ability of selen chelate pea oligopeptide and its synthesis raw materials in Example 1 of the present invention. [Figure 14] This is a comparison chart of VC reduction capabilities. [Figure 15] This is the primary mass spectral diagram of the peptide fragment TGRGAP. [Figure 16] This is a secondary mass spectral diagram of the peptide fragment TGRGAP. [Figure 17] This is the primary mass spectrum diagram of the peptide fragment PPKIYP. [Figure 18] This is a secondary mass spectral diagram of the peptide fragment PPKIYP. [Figure 19] This is the primary mass spectrum diagram of the peptide fragment HQMPKP. [Figure 20] This is a secondary mass spectral diagram of the peptide fragment HQMPKP. [Figure 21] This is the primary mass spectral diagram of the peptide fragment TSSLP. [Figure 22] This is a secondary mass spectral diagram of the peptide fragment TSSLP. [Figure 23] This is a mass spectral diagram of the synthetic peptide fragment TGRGAP standard. [Figure 24]This is a mass spectral diagram of the synthetic peptide fragment PPKIYP standard. [Figure 25] This is a mass spectral diagram of the synthetic peptide fragment HQMPKP standard. [Figure 26] This is a mass spectral diagram of the synthetic peptide fragment TSSLP standard. [Figure 27] This figure shows the effect of temperature on the chelation reaction in Experimental Example 1. [Figure 28] This figure shows the effect of the mass ratio of the peptide salt on the chelation reaction in Experimental Example 2. [Figure 29] This figure shows the effect of pea oligopeptide concentration on the chelation reaction in Experimental Example 3. [Figure 30] This figure shows the effect of pH value on the chelation reaction in Experimental Example 4. [Figure 31] This figure shows the effect of reaction time on the chelation reaction in Experimental Example 5. [Modes for carrying out the invention]

[0040] The following will clearly and completely describe the technical solutions in embodiments of the present invention with reference to the drawings relating to embodiments of the present invention. Naturally, the embodiments described are only a part of the embodiments of the present invention and not all embodiments.

[0041] The raw material information used in the following examples and experimental cases is listed below. Pea oligopeptide: Manufactured in-house. Sodium selenite: Analytically pure, purchased from Tianjin Damao Chemical Reagent Plant. 3'3-Diaminobenzidine (DAB 4HCl): Reagent grade. Disodium ethylenediaminetetraacetate (EDTA-2Na): Biotechnology grade, manufactured by Biotopped Amresco. Hydrobromic acid: Analytically pure, manufactured by Tianjin Fuchen Chemical Reagent Plant. Hydroxylamine hydrochloride, perchloric acid, nitric acid, hydrochloric acid, sodium hydroxide, 95% ethanol, toluene, trichloroacetic acid: Analytically pure, all purchased from Beijing Chemical Plant. Pepsin, trypsin: Manufactured by Nanning Pangbo Biotechnology Co., Ltd. Trifluoroacetate: Analytically pure, manufactured by Alfa Aesar. Acetonitrile: Chromatographically pure, manufactured by Fisher. 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH): Manufactured by Sigma, Inc., USA. Ultrapure water: It is produced in-house in the laboratory.

[0042] The pea oligopeptide was specifically prepared using the following process. Pea protein powder (with a protein content of approximately 82%) is mixed with pure water in a ratio of approximately 1:10, and after stirring thoroughly, food-grade sodium hydroxide is added to adjust the pH of the liquid to approximately 9, the temperature is controlled to 50±2℃, and 2.4L of Alcalase is added. (2 0.5% , w / w) and Neutrase 0.8L ( 1.5% , w / w) (Both types of proteases are purchased from Novozymes) are added, and the enzymatic decomposition time is approximately 4.5 hours. After the enzymatic decomposition time is complete, the material liquid is heated to 120±2℃ using a plate heat exchanger for approximately 20 seconds to inactivate the enzymes. The material liquid is then centrifuged, filtered through a ceramic membrane, vacuum concentrated, sterilized, and then sprayed and dried into a powder to produce pea oligopeptide powder.

[0043] Using the high-performance gel filtration chromatography method described in the reference "In vitro antioxidant effects of corn oligopeptides [J]. Food Science, 2011, 32(5):22-26.", the molecular weight and distribution of pea oligopeptides were determined by the percentage of peak area. Here, the proportion of components with a molecular weight > 5000u was 0, the proportion of components with a molecular weight of 2000-5000u was 0.95%, the proportion of components with a molecular weight of 1000-2000u was 7.79%, the proportion of components with a molecular weight of 500-1000u was 25.25%, the proportion of components with a molecular weight of 140-500u was 60.50%, and the proportion of components with a molecular weight < 140u was 5.51%. Calculations show that 91.26% of the components have a molecular weight less than 1000u.

[0044] The equipment used in the following examples and experiments is listed below. EL20 pH meter: Manufactured by Mettler Toledo. KQ-250E ultrasonic oscillator: Manufactured by Kunshan Ultrasonic Instruments Co., Ltd. 1204007 constant temperature water bath: Manufactured by Suzhou POXIWAR Laboratory Equipment Co., Ltd. Microplate reader: Manufactured by Dynex Spectra Mr. DHG-9075A electric heating constant temperature blast drying oven: Manufactured by Beijing Luxi Technology Co., Ltd. Universal electric furnace: Manufactured by Beijing Kewei Yongxing Instruments Co., Ltd. LC-20AD high-performance liquid chromatograph: Manufactured by Shimadzu Corporation. F30200150 Kjeldahl device: Manufactured by Velp Scientifica.

[0045] In the following examples and experimental cases, Selenium Chelate Pea Oligopeptide The methods for detection and evaluation are listed below. 1. Molecular weight distribution measurement The measurement is performed using a high-performance gel filtration chromatography method. Five peptide standards, such as glycine-glycine-glycine (molecular weight 189), glycine-glycine-tyrosine-arginine (molecular weight 451), bacillus enzyme (molecular weight 1450), aprotinin (molecular weight 6500), and cytochrome C (molecular weight 12500), are selected and prepared as 0.1% (M / V) solutions. The samples are filtered using a polytetrafluoroethylene filter membrane with a pore size of 0.2 μm, and the samples are injected. Using high-performance liquid chromatography, gel filtration is performed. Create a relative molecular weight calibration curve. do. The mobile phase was V(acetonitrile):V(water):V(trifluoroacetate) = 45:55:0.1, the sample injection volume was 10 μL, the flow rate was 0.5 mL / min, the detection wavelength was 220 nm, the column temperature was 30 °C, and detection was performed using an ultraviolet detector. The data was processed using GPC software. By substituting the chromatographic data of the sample into the calibration curve equation, the molecular weight of the peptide in the sample and its distribution range can be obtained. The relative percentage of peptides with different molecular weight ranges can be calculated using the peak area normalization method.

[0046] 2. Measurement of selenium content Accurately weigh 2.190 g of Na2SeO3·5H2O, dissolve it in a small amount of ultrapure water, add 48% hydrobromic acid, and then adjust the volume to 1 L with ultrapure water to prepare a 657.4746 mg / L selenium standard stock solution.

[0047] Aspirate 1 mL of the stock selenium standard solution, and adjust the volume to 100 mL with ultrapure water to prepare a 6.57 μg / mL selenium standard working solution.

[0048] Accurately measure 0, 2, 4, 6, 8, and 10 mL of selenium standard working solution. After decomposition with acid, measure the absorbance at 420 nm of the yellow complex produced after the reaction of 3,3'-diaminobenzidine with reduced selenium. For specific procedures, refer to the spectrophotometric method proposed in the literature "Spectrophotometric Determination of Selenium by 3,3'-Diaminobenzidine [C]. Representative Conference and Annual Scientific Symposium of the Branch of Veterinary Internal Medicine, Chinese Society of Animal Husbandry and Veterinary Medicine. 2011."

[0049] As shown in Figure 1, the selenium standard curve is drawn with selenium concentration as the x-coordinate and absorbance as the y-coordinate.

[0050] A fixed amount of the sample to be measured is accurately weighed, dissolved in 10 mL of ultrapure water, and then the sample is treated using the method described above. The absorbance of the treated sample is then measured. The selenium content is determined in accordance with the standard curve.

[0051] 3. Chelation rate and yield The formula for calculating the chelation rate is: Chelation rate (%) = m1 ÷ m2 × 100%, where m1 is Selenium Chelate Pea Oligopeptide Selenium content ( Selenium Chelate Pea Oligopeptide m2 is the mass of selenium in sodium selenite, and m2 is the mass of selenium in sodium selenite. The formula for calculating the chelate yield is: The yield (%) is calculated as m3 ÷ m4 × 100%, where m3 is the mass of the chelated product and m4 is the total mass of the substance to be added to the chelation system.

[0052] 4. Measurement of basic physical and chemical properties The National Standard Law GB 5009.3-2010 was adopted. Selenium Chelate Pea Oligopeptide The moisture content is measured. The national standard GB 5009.5-2010 is adopted. Selenium Chelate Pea Oligopeptide Measure the total nitrogen (protein) content. Refer to National Standards Law GB 22729-2008. Selenium Chelate Pea Oligopeptide The acid-soluble protein content is measured.

[0053] 5. Thermal stability experiment Selenium Chelate Pea Oligopeptide The sample was dissolved in ultrapure water to prepare a solution with a concentration of 2 mg / mL. 30 mL of each solution was placed in a centrifuge tube and subjected to constant temperature water baths at 25, 40, 60, 80, and 100°C for 2 hours, with 25°C serving as the room temperature control group. After cooling to room temperature, the molecular weight distribution was detected, and a 3 mL sample was placed in a dialysis bag and dialyzed for 60 hours to detect the selenium content.

[0054] 6. Acid-base stability experiment Selenium Chelate Pea Oligopeptide The sample is dissolved in ultrapure water to prepare a solution with a concentration of 2 mg / mL. 30 mL of the solution is taken and placed in a centrifuge tube. The pH of each tube is adjusted to 3, 5, 7, 9, and 11 using 1 mol / L HCl and 1 mol / L NaOH, respectively. The tubes are left in a 37°C constant temperature water bath for 2 hours. Simultaneously, one untreated control group is set up. After cooling to room temperature, the molecular weight distribution is detected. A 3 mL sample is taken and placed in a dialysis bag, and the selenium content is detected by dialysis for 60 hours.

[0055] 7. In vitro simulation experiment of the gastrointestinal tract 7.1 Pepsin digestion experiment Selenium Chelate Pea Oligopeptide Dissolve the sample in ultrapure water to prepare a solution with a concentration of 2 mg / mL. Take 40 mL of this solution and place it in a centrifuge tube. Adjust the pH of the solution to 2 with 1 mol / L HCl and preheat in a 37°C water bath for 20 mins. Then, add 6‰ (relative to the material) of pepsin, mix thoroughly, quickly remove 20 mL and place it in another centrifuge tube. Treat in a 100°C boiling water bath for 10 mins to prepare a pre-digestion control. Treat the remaining portion in a 37°C water bath for 4 hours, and then inactivate the enzyme in a 100°C boiling water bath. After cooling to room temperature, detect the molecular weight distribution, take a 3 mL sample, place it in a dialysis bag, and dialyze for 60 hours to detect the selenium content.

[0056] 7.2 Trypsin digestion experiment Selenium Chelate Pea OligopeptideDissolve the sample in ultrapure water to prepare a solution with a concentration of 2 mg / mL. Take 40 mL of the solution and place it in a centrifuge tube. Adjust the solution pH to 7.5 with 1 mol / L NaOH and preheat in a 37°C water bath for 20 mins. Add 2‰ (relative to the material) of trypsin, mix uniformly, then quickly remove 20 mL and place it in another centrifuge tube. Treat in a 100°C boiling water bath for 10 mins to prepare a pre-digestion control. Treat the remaining portion in a 37°C water bath for 6 hours, then inactivate the enzyme in a 100°C boiling water bath. After cooling to room temperature, detect the molecular weight distribution. Take a 3 mL sample, place it in a dialysis bag, and dialyze for 60 hours to detect the selenium content.

[0057] 7.3 Pepsin digestion experiment first, then trypsin digestion experiment After pepsin digestion according to the method for pepsin digestion experiment in 7.1, the pH is adjusted to 6.8 with 1 mol / L NaOH, preheated in a 37°C constant temperature water bath for 20 mins, 2‰ (relative to the material) of trypsin is added and mixed uniformly, then 20 mL is quickly removed and placed in another centrifuge tube, and treated in a 100°C boiling water bath for 10 mins to be used as a pre-digestion control. The remaining portion is treated in a 37°C constant temperature water bath for 6 hours, then the enzyme is inactivated in a 100°C boiling water bath, cooled to room temperature, and the molecular weight distribution is detected. A 3 mL sample is placed in a dialysis bag and dialyzed for 60 hours to detect the selenium content.

[0058] 8. Antioxidant function evaluation 8.1 Measurement of DPPH free radical scavenging capacity Samples of different concentrations were taken and mixed with a 0.1 mol / L DPPH-absolute ethanol solution in a 1:1 volume ratio. The mixture was stored in the dark for 30 minutes, and the absorbance of the mixture was measured at 517 nm in the ultraviolet-visible spectrum. i It is noted that, accordingly, sample solutions of different mass concentrations were uniformly mixed with absolute ethanol solution in a 1:1 volume ratio, stored in the dark at room temperature for 30 minutes, and the absorbance was measured at 517 nm in the ultraviolet-visible spectrum. jIt is noted that distilled water is uniformly mixed with 0.1 mol / L DPPH-absolute ethanol solution in a 1:1 volume ratio, stored in the dark at room temperature for 30 minutes, and the absorbance is measured at 517 nm in the ultraviolet-visible spectrum. c The following is noted. Each experiment is repeated three times, the mean is taken, and the standard deviation is calculated. Sodium selenite and pea oligopeptide are used as raw material controls before chelation, and ascorbic acid is used as a positive control. The removal rate of DPPH free radicals from the sample is calculated according to the following formula.

number

[0059] 8.2 Measurement of OH free radical removal capacity Take sample solutions of different concentrations and uniformly mix them with 5 mol / L FeSO4 and 5 mol / L salicylic acid-absolute ethanol solution in a volume ratio of 1:2:2. Start the reaction with 1 volume of 5 mol / L H2O2 solution and react in a 37°C water bath for 1 hour. Measure the absorbance at 510 nm in the UV-Vis spectrum and label it A2. Correspondingly, replace the 5 mol / L H2O2 solution with 1 volume of water, keeping the ratios of the other reagents unchanged, react in a 37°C water bath for 1 hour, measure the absorbance at 510 nm in the UV-Vis spectrum and label it A20. Replace the sample solution with distilled water, keeping the ratios of the other reagents unchanged, react in a 37°C water bath for 1 hour, measure the absorbance at 510 nm and label it A 02 The following is noted. Each experiment is repeated three times, the mean is taken, and the standard deviation is calculated. Sodium selenite and pea oligopeptide are used as raw material controls before chelation, and ascorbic acid is used as a positive control. The removal rate of OH free radicals from the sample is calculated according to the following formula.

number

[0060] 8.3 Measurement of reducing ability Sample solutions of different concentrations are taken and uniformly mixed with 0.2 mol / L phosphate buffer (pH 6.6) and 1% K3[Fe(CN)6] solution in a 1:1:1 volume ratio. The mixture is stored in a 50°C water bath for 10 minutes, then rapidly cooled with cold water. A 10% trichloroacetic acid solution is added at a 1:1 volume ratio, and the mixture is shaken quickly, thoroughly, and uniformly in the dark. A 1:1 volume ratio reaction mixture is then taken out, and a 1:1 volume ratio of distilled water and a 0.2 volume ratio of 0.1% FeCl3 solution are added. The mixture is shaken thoroughly and uniformly in the dark, and then allowed to stand for 10 minutes. The absorbance is measured at 700 nm and recorded as A3. Sodium selenite and pea oligopeptide are used as the raw material controls before chelation, and ascorbic acid is used as the overall control. The absorbance A3 is directly used as the sample reducing ability parameter. Each experiment is repeated three times, the mean is taken, and the standard deviation is calculated.

[0061] 9. Identification of peptide fragments and identification of antioxidant capacity

[0062] 9.1 Peptide Fragment Identification (1) Setting up LC-MS / MS Peptide fragments are analyzed using liquid-phase chromatography-tandem mass spectrometry and separated by setting the liquid-phase chromatography parameters as follows.

[0063] The sample concentration was 5 mg / mL, and gradient elution was performed for 30 minutes using an Ultimate 3000 HPLC liquid-phase system connected to a Q Exactive mass spectrometer (LC-MS / MS, Thermo Scientific) at a flow rate of 0.30 μL / min. The analytical column was a custom-made quartz capillary (inner diameter 75 μm, column length 15 cm, Upchurch, Oak Harbor, WA) containing C18 packing (300 Å, 5 μm, Varian, Lexington, MA). Mobile phase A was 0.1% formic acid solution, and mobile phase B contained 80% acetonitrile and 0.1% formic acid.

[0064] Q Exactive mass spectrometry uses the Xcalibur 2.1.2 software data-dependent acquisition method, with a primary detection type of orbit trap, a primary full scan mass range of 100-1200 m / z, a resolution of 70,000, and secondary fragmentation performed by selecting the first 20 ions in order of signal intensity, with a secondary detection type of ion trap, a resolution of 17,500, and a collision energy of 27%.

[0065] (2) Data processing The raw mass spectrometry data is obtained using de novo sequencing of the database and the peptide fragment, respectively. Suspicious peptide fragments obtained from the database are compared with suspicious peptide fragments obtained by searching the database using PEAKS.

[0066] The database was searched using Proteome Discovery software (Version PD1.4, Thermo-Fisher Scientific, USA) with the following search criteria: no enzymatic digestion, dynamic modification including oxidation (M), precursor ion mass deviation set to 20 ppm, and secondary mass deviation set to 0.02 Da. The FDR value was calculated using Percolator in the PD library, and the FDR indicates the peptide fragment false positive rate. A q value less than 1% can be considered correct for peptide spectral matching. A peptide fragment designated as a specific protein can be considered unique. For protein recognition, the false positive rate is set to 0.01.

[0067] (3) Peptide fragment synthesis Peptide fragment standards are provided by GL Biochem (Shanghai) Ltd. The peptide fragment synthesis route is as follows: FMOC-ARG(PBF)-WANG RESIN resin selection → removal of FMOC from the resin with hexahydropyridine → introduction of the second amino acid FMOC-CYS(TRT)-OH reaction using HOBT and DIC as condensing agents → after complete reaction, subsequent removal of FMOC, linking to the next amino acid, and the process is repeated until the last amino acid is linked → processing of the resin using TFA solution to obtain the crude peptide product → dissolving the crude polypeptide product, adding glutathione in an appropriate ratio, stirring at low temperature, and transferring to MS at 6-12 hour intervals to check the oxidation state, while simultaneously using a sulfhydryl detection agent to help determine if it is completely oxidized → after complete oxidation, purification to obtain the standard synthetic peptide fragment (synthetic peptide fragment standard).

[0068] 9.2 Identification of Peptide Fragment Antioxidant Properties Using the ABTS+ method Selenium Chelate Pea Oligopeptide The antioxidant properties of the peptide fragments were identified, and a standard curve was drawn using Trolox as an antioxidant standard. The prepared standard synthetic peptide fragments (5 mg / mL) and Selenium Chelate Pea Oligopeptide Each of the 5 mg / mL solutions was reacted using the ABTS kit (Biyuntian), and its absorbance was measured at 734 nm to investigate its antioxidant capacity.

[0069] 9.3 Quantitative Analysis of Peptide Fragments Selenium Chelate Pea Oligopeptide Quantitative analysis was performed on the peptide fragment, and the specific instrument conditions were as follows.

[0070] (1) Instrument and reagent information The instrumentation is HPLC-MS / MS (LC liquid phase is from DIONEX, and Ultimate3000-MS mass spectrometry is an API 3200 Q TRAP from AB Corporation in the USA), and methanol, nitriles, etc., are all purchased from Fisher.

[0071] (2) Sample pretreatment Add an appropriate amount of distilled water and an equal volume of protein precipitant (acetonitrile, containing an internal standard of 100 ng / mL), vortex for 2 minutes, apply sonication for 2 minutes, centrifuge at 13200 rpm for 4 minutes, and take the supernatant for measurement.

[0072] (3) Liquid phase conditions: Chromatography column: MSLAB HP-C18 (150*4.6mm 5μm 120a), column temperature: 50℃, flow rate: 1mL / min. Mobile phase: A. Aqueous phase: Water (2MMOL / L ammonium formate), B. Organic phase: Acetonitrile (2MMOL / L ammonium formate). Sample injection volume: 10 μL. Gradient: 0-2 min: 95% A + 5% B, 2.1-5.0 min: 20% A + 80% B, 5.1-7 min: 0% A + 100% B, 7.1-10 min: 95% A + 5% B.

[0073] (4) Mass spectrometry conditions TIFF0007855350000003.tif73170

[0074] 10. Statistical Processing Statistical analysis of experimental data was performed using SPSS 13.0 software, and a t-test was used to compare the pairs. If P < 0.05, the difference between the two groups is significant.

[0075] Example 1

[0076] Dissolve 5g of pea oligopeptide in 100mL of ultrapure water to prepare a 5g / 100mL aqueous solution of pea oligopeptide. Add 2.5g of sodium selenite and mix well with an ultrasonic oscillator. Adjust the pH to 9.0 and react for 30 minutes under conditions of a constant temperature water bath at 80°C. While still hot... ultrapure water Pour in four times the volume of 95% ethanol. Let stand overnight. Discard the supernatant and dry the remaining portion in a 35°C constant temperature blast drying oven. Selenium Chelate Pea Oligopeptide To obtain.

[0077] Example 2

[0078] Dissolve 4g of pea oligopeptide in 100mL of ultrapure water to prepare a 4g / 100mL aqueous solution of pea oligopeptide. Add 2g of sodium selenite and mix well with an ultrasonic oscillator. Adjust the pH to 8.0 and react for 30 minutes under conditions of a constant temperature water bath at 85°C. While still hot... ultrapure water Pour in four times the volume of 95% ethanol. Let stand overnight. Discard the supernatant and dry the remaining portion in a 35°C constant temperature blast drying oven. Selenium Chelate Pea Oligopeptide To obtain.

[0079] Example 3

[0080] Dissolve 5g of pea oligopeptide in 100mL of ultrapure water to prepare a 5g / 100mL aqueous solution of pea oligopeptide. Add 1.67g of sodium selenite and mix well with an ultrasonic oscillator. Adjust the pH to 8.0 and react for 30 minutes under conditions of a constant temperature water bath at 90°C. While still hot... ultrapure water Pour in four times the volume of 95% ethanol and let stand overnight. Discard the supernatant and dry the remaining portion in a 35°C constant temperature blast drying oven. Selenium Chelate Pea Oligopeptide To obtain.

[0081] Example 4

[0082] Dissolve 3g of pea oligopeptide in 100mL of ultrapure water to prepare a 3g / 100mL aqueous solution of pea oligopeptide. Add 1.5g of sodium selenite and mix well with an ultrasonic oscillator. Adjust the pH to 8.5 and react for 30 minutes under conditions of a constant temperature water bath at 90°C. While still hot... ultrapure water Pour in four times the volume of 95% ethanol and let stand overnight. Discard the supernatant and dry the remaining portion in a 35°C constant temperature blast drying oven. Selenium Chelate Pea Oligopeptide To obtain.

[0083] In the above examples 1 to 4 Selenium Chelate Pea OligopeptideThe basic physical and chemical properties are shown in Table 1. As can be seen from Table 1, the results obtained by Examples 1-4 Selenium Chelate Pea Oligopeptide They have similar basic physical and chemical properties, both contain over 23% acid-soluble protein, both have over 23% total nitrogen, and acid-soluble protein accounts for over 97% of the crude protein, which means that Selenium Chelate Pea Oligopeptide This indicates that the molecular weight of the proteins in this sample is low, which is advantageous for human absorption.

[0084] In Examples 1 to 4, Selenium Chelate Pea Oligopeptide The moisture content of all of them is within the range of 14.17% ± 1.12%. This moisture content is equivalent to that of wheat flour with a moisture content of 15-17%, and the corresponding aw value is 0.80-0.87, which inhibits most molds and staphylococci, making storage easy.

[0085] As shown in Table 1, in Examples 1 to 4, Selenium Chelate Pea Oligopeptide All of them have a selenium content of 0.08g / 100g or more, indicating a high selenium content. Selenium Chelate Pea Oligopeptide It is proven that the product can be obtained. Further calculations using the methods described in Examples 1 to 4 show that Selenium Chelate Pea Oligopeptide The chelation rate can reach 20% or more, and even 50% or more, and at the same time, Selenium Chelate Pea Oligopeptide The yield can be 10% or more, and even reach 30% or more.

[0086] [Table 1]

[0087] The results obtained by preparing in Examples 1 to 4 Selenium Chelate Pea Oligopeptide Table 2 shows the specific molecular weight distribution. Here, in Example 1 Selenium Chelate Pea Oligopeptide The molecular weight distribution gel chromatogram is shown in Figure 2.

[0088] As can be seen from Table 2 and Figure 2, more than 85% of the molecular weight distribution consists of molecules with a molecular weight of 1000u or less, with the highest content being small peptides with a molecular weight of <500u, accounting for more than 68%. This indicates that these types of small peptides are more functional in living organisms.

[0089] [Table 2]

[0090] The result obtained in Example 1 Selenium Chelate Pea Oligopeptide Using the sample, the following tests, characterizations, and evaluations were performed, including the following:

[0091] 1. Ultraviolet full-wavelength scanning Aqueous solution of pea oligopeptide and Selenium Chelate Pea Oligopeptide Each aqueous solution was prepared at a concentration of 0.05 g / mL, and a full-wavelength ultraviolet scan was performed. The scanning wavelength range was 200-600 nm, and the results are shown in Figure 3.

[0092] As can be seen from Figure 3, the pea oligopeptide has a maximum absorption peak at 309 nm, and the pea oligopeptide reacts with sodium selenite. Selenium Chelate Pea Oligopeptide When this structure is formed, the maximum absorption peak is redshifted to 322 nm, and the OD value of the maximum absorption peak also increases. This demonstrates that a change occurs in the material structure of the pea oligopeptide, and that the combination of selenium and the peptide forms a structure with stronger light absorption performance, which is a result of a change in the degree of valence electron transition after the reaction between the pea oligopeptide and selenium.

[0093] 2. Photographs taken with a scanning electron microscope. Figures 4(a) and 4(b) show pea oligopeptides and Selenium Chelate Pea Oligopeptide These are scanning electron microscope images. As can be seen from Figure 4(a), before the reaction, the pea oligopeptides are clearly spherical granules, and at 1000x magnification, folds are present on the surface of the pea oligopeptide particles, and the particle size of all perfect particles is smaller than 50 μm. As can be seen from Figure 4(b), after the reaction, Selenium Chelate Pea Oligopeptide The shape of the particles changed significantly and they lost their spherical morphology. Most big have a particle size larger than 300 μm. Selenium Chelate Pea Oligopeptide Most of the surface wrinkles of have unfolded and there are holes. There are obvious changes in the particles from front to back, proving that they are two kinds of substances. It is speculated that through the chelation reaction, the surface of the pea oligopeptide unfolds and its structure changes, thereby providing binding sites for selenium.

[0094] 3. Fourier transform infrared spectroscopy As shown in Figure 5, by comparing the absorption peaks of pea oligopeptide and Selenium Chelate Pea Oligopeptide (chelta), the shape of the peaks changed. Specifically, the absorption peak of pea oligopeptide is at 3040 cm -1 , 954 cm -1 附近, which proves the existence of -COOH. However, Selenium Chelate Pea Oligopeptide has a narrower absorption peak near 3040 cm -1 and becomes very weak near 954 cm -1 . This proves that there is no free carboxyl group and the carboxyl group may be bonded to selenium in the form of a covalent bond. Selenium Chelate Pea Oligopeptide has double peaks near 3158 cm -1 and 3436 cm -1 , and a peak near 1216 cm -1 . This proves the existence of -NH2 and that -NH2 is a free radical.

[0095] Analyzing theoretically, Selenium Chelate Pea Oligopeptide 's chelation mechanism is that Se 4+ binds with -NH2, and the carboxyl group also binds with Se 4+ by covalent bond. In the pea oligopeptide selenium chelate, Se 4+It is hypothesized that a 4d empty orbital is provided by O and N, and that lone pairs of electrons are provided by O and N, allowing them to occupy the empty orbitals and form a coordination bond. This hypothesis is consistent with other related studies, and you may refer to the following references specifically: "Gao Fei, Wang Weiyou, Lu Jun, et al., Preparation and Infrared Spectroscopic Characterization of Marine Fish Collagen Peptide Calcium Chelate [J]. Journal of Ocean University of China (Natural Science Edition), 2015, 45(1):47-54." and "Song Shasha, Gao Fei, Ren Difeng, et al., Preparation and Infrared Spectroscopic Identification of Black Bone Chicken Peptide Iron(II) Chelate [J]. Food and Fermentation Industry, 2013, 39(6):13-17."

[0096] 4. Selenium Chelate Pea Oligopeptide Thermal stability Selenium Chelate Pea Oligopeptide The molecular weight distribution is shown in Table 3 after treatment at 40, 60, 80, and 100°C for 2 hours. As can be seen from Table 3, after heat treatment, Selenium Chelate Pea Oligopeptide The proportion of molecules with a molecular weight of less than 1000u fluctuates around 93%, while the range of change in the proportion of molecules with a molecular weight greater than 1000u is less than 2%.

[0097] [Table 3]

[0098] Overall, the proportion of 140-1000u increased due to the influence of temperature. After processing at different temperatures, Selenium Chelate Pea Oligopeptide The selenium content is shown in Figure 6. Compared to the control, Selenium Chelate Pea Oligopeptide No significant difference in selenium content was observed after treatment at different temperatures (a=0.05). Selenium Chelate Pea Oligopeptide It has been proven to have excellent thermal stability. This is likely because pea oligopeptides only possess the primary structure of a protein and therefore have strong resistance to temperature changes.

[0099] 5. Selenium Chelate Pea Oligopeptide Acid-base stability Table 4 shows the molecular weight distribution of pea oligopeptide selenium chelate after treatment under different pH conditions. Under neutral conditions, the proportion of molecules with a molecular weight of less than 140u increased by approximately 10%, showing the largest change in this category. This may be because these types of small peptides (especially those with a molecular weight of less than 500u) are resistant to acid-base environments but are easily destroyed under neutral conditions. Molecules with a molecular weight of less than 1000u did not change significantly in proportion, with the maximum change not exceeding 3%. Furthermore, this can be seen as demonstrating the resistance of the peptide chain to acid-base environments.

[0100] [Table 4]

[0101] Figure 7 shows the change in selenium content after treatment at different pH levels. In nearly acidic and neutral environments, the selenium content decreases significantly (a=0.05), with a change rate of approximately 20%. In an alkaline environment, the selenium content changes only slightly. However, the fact that the selenium content remains above 78% within the pH range of 3 to 11 demonstrates that the chelated product of pea oligopeptide and selenium has strong stability in acidic and basic environments.

[0102] 6. Selenium Chelate Pea Oligopeptide In vitro digestive stability Selenium Chelate Pea Oligopeptide Each sample was treated with pepsin and trypsin, respectively, with the first sample treated with pepsin and the second with trypsin. The molecular weight distribution results are shown in Table 5. As can be seen from Table 5, Selenium Chelate Pea Oligopeptide After being processed by different in vitro digestion methods, the proportion of molecules with a molecular weight of less than 1000u increased, and here, after being processed by pepsin... Selenium Chelate Pea Oligopeptide Its molecular weight increased by approximately 7% and it was treated with trypsin. Selenium Chelate Pea Oligopeptide It increased by approximately 9%, and was first treated with pepsin and then with trypsin. Selenium Chelate Pea OligopeptideThis increased by approximately 12%. This demonstrates that pea oligopeptides were digested by enzymes and broken down into smaller molecular weight peptide fragments. After enzymatic digestion, the proportion of molecules under 1000u was over 90% in all cases, indicating that small molecule peptide fragments, especially dipeptides and tripeptides, are favored for human absorption.

[0103] As shown in Figure 8, after processing using different digestion methods, Selenium Chelate Pea Oligopeptide The change in selenium content was small, and in all cases, it was less than 3% compared to the change before digestion. Selenium Chelate Pea Oligopeptide Referring to the molecular weight distribution change table, it can be inferred that although the selenium oligopeptide is broken down into small peptide fragments by hydrolysis after enzymatic digestion, the coordination structure between selenium and the peptide is not completely destroyed. Therefore, the coordination bond between pea oligopeptide and selenium is relatively stable and is not destroyed by the cleavage of the peptide chain. Selenium, along with the small peptide fragments, can stably pass through gastric and intestinal juices and is finally absorbed indirectly in the intestines by absorption by the peptide on the small intestinal mucosa.

[0104] [Table 5]

[0105] 7. Removal of DPPH free radicals from pea oligopeptide selen chelate In Figure 9, ■, ●, and ▲ represent sodium selenite, pea oligopeptide, and respectively. Selenium Chelate Pea Oligopeptide This is representative of the case. As shown in Figure 9, sodium selenite has a weak ability to remove DPPH free radicals, and its removal rate remains stable at approximately 20% regardless of the concentration of sodium selenite.

[0106] Pea oligopeptides have the ability to remove DPPH free radicals, and the removal rate increases with increasing pea oligopeptide concentration, exhibiting a parabolic trend. The rate of removal gradually decreases, and the IC of pea oligopeptides for removing DPPH free radicals follows a parabolic equation.50 The value is calculated to be 3.39 ± 0.02 mg / mL.

[0107] Selenium Chelate Pea Oligopeptide Its ability to remove DPPH free radicals is relatively strong and changes with the chelate dose. As the chelate concentration increases, its removal rate of DPPH free radicals also gradually increases. Within a concentration range of less than 5 mg / mL, the trend in the removal rate is almost linear, and as the concentration continues to increase, the rate of change in removal gradually slows down, exhibiting a parabolic trend.

[0108] By calculation, Selenium Chelate Pea Oligopeptide IC 50 = 1.77 ± 0.01. After chelation, the chelate's ability to remove DPPH free radicals becomes stronger than that of the raw materials, pea oligopeptide and sodium selenite, and its IC 50 The value is approximately half that of pea oligopeptide. Calculated according to Figure 10, the IC of positive control ascorbic acid is... 50 The value is 4.85 ± 0.02 μg / mL.

[0109] 8. Removal of OH free radicals from pea oligopeptide selen chelate In Figure 11, ■, ●, and ▲ represent sodium selenite, pea oligopeptide, and respectively. Selenium Chelate Pea Oligopeptide This is representative. As shown in Figure 11, the removal capacity for OH free radicals changes almost linearly with increasing slope as the sodium selenite concentration increases. When the sodium selenite concentration is 5 mg / mL, the removal rate for OH free radicals can reach 100%. The IC of sodium selenite for OH free radical removal follows the fitting curve equation. 50 This is obtained by calculating = 1.23 ± 0.02 mg / mL.

[0110] Pea oligopeptides have a certain ability to remove OH free radicals, and the removal rate shows a parabolic trend as the pea oligopeptide concentration increases. According to the fitted parabolic equation, the IC of OH free radical removal by pea oligopeptides is... 50 The value was calculated to be 23.55 ± 0.07 mg / mL.

[0111] Selenium Chelate Pea Oligopeptide It has a strong ability to remove OH free radicals, as can be seen in Figure 11. In the range of concentrations less than 5 mg / mL, the removal rate is close to a linear curve with a steep slope as the concentration changes, but as the concentration continues to increase, the change in the removal curve tends to become gentler, the slope becomes smaller, and it is almost the same as the change in slope of pea oligopeptide. In the range of concentrations less than 5 mg / mL, Selenium Chelate Pea Oligopeptide The removal capacity of sodium selenite against OH free radicals is greatly affected by the sodium selenite concentration, but beyond this range, the removal capacity of sodium selenite against OH free radicals becomes saturated. Selenium Chelate Pea Oligopeptide It is presumed that the free radical removal capacity is mainly influenced by the concentration of the pea oligopeptide, which is the raw material. According to the fitting curve Selenium Chelate Pea Oligopeptide IC removes OH free radicals. 50 The value was calculated to be 3.28 ± 0.04 mg / mL. According to Figure 12, the IC2000 ion (IC2000) removes OH free radicals from positive control ascorbic acid. 50 The value is 1024.87 ± 5.96 μg / mL.

[0112] 9, Selenium Chelate Pea Oligopeptide Reduction capacity In Figure 13, ■, ●, and ▲ represent sodium selenite, pea oligopeptide, and respectively. Selenium Chelate Pea Oligopeptide This is representative of the results. As shown in Figure 13, sodium selenite has no reducing ability. The reducing ability of pea oligopeptides increases with increasing concentration, and the change shows a linear trend. Selenium Chelate Pea Oligopeptide The reducing capacity also increases with increasing concentration, and the change tends to be close to a linear pattern, Selenium Chelate Pea OligopeptideIts reducing ability is higher than that of the pea oligopeptide used as its raw material.

[0113] As shown in Figure 14, the reducing capacity of positive control ascorbic acid exhibits a parabolic trend with increasing concentration. Within the range of ascorbic acid concentrations below 200 μg / mL, the curve becomes nearly linear, and as the concentration gradually increases, the rate of change in reducing capacity gradually decreases. If the absorbance corresponding to the reducing capacity of ascorbic acid is approximately 1.6, the required concentration of ascorbic acid is 400 μg / mL, and accordingly, to reach this absorbance, the required concentration is... Selenium Chelate Pea Oligopeptide The concentration is 50 mg / mL. When pea oligopeptide is at a concentration of 50 mg / mL, it can only achieve an absorbance of approximately 0.8. From Figure 13, Selenium Chelate Pea Oligopeptide It can be inferred that the improvement in reducing ability is not due to a simple superposition of the raw materials, pea oligopeptide and sodium selenite, but rather to an enhancement of reducing ability through chelation techniques.

[0114] 10, Selenium Chelate Pea Oligopeptide Identification of peptide fragments and antioxidant capacity Selenium Chelate Pea Oligopeptide To accurately identify possible peptide fragments, the results identified by the database and the PEAKS database search results are compared to find possible peptide fragments. and , by de novo method same The defined peptide fragment and of We will analyze and compare the samples, select four reliable peptide fragments, and then perform mass spectrometry structural identification.

[0115] Refer to Figures 15 to 22 for the primary and secondary mass spectra of the four selected reliable peptide fragments, Figures 23 to 26 for the mass spectra of the four standard synthetic peptide fragments, and Table 6 for the results of mass spectrometry structural identification and quantitative analysis of the standard synthetic peptide fragments and Selenium Chelate Pea Oligopeptide See Table 7 for the results of identifying its antioxidant properties.

[0116] Table 7 shows that, Selenium Chelate Pea Oligopeptide It has relatively strong antioxidant capacity. Of the four peptide fragments identified by mass spectrometry, PPKIYP is Selenium Chelate Pea Oligopeptide It is a peptide fragment with relatively strong antioxidant capacity.

[0117] [Table 6]

[0118] [Table 7]

[0119] Experimental Example 1: Effect of reaction temperature on chelation results The pea oligopeptide aqueous solution was prepared at a concentration of 1% (i.e., 1 g / 100 mL), with a mass ratio of pea oligopeptide to sodium selenite (mass ratio of peptide salts) of 2:1, and a pH of 8.5. The reaction was carried out for 30 minutes at 60, 70, 80, 85, and 90°C, respectively, and the effect of reaction temperature on the yield and chelation rate was investigated.

[0120] As can be seen from Figure 27, temperature has a significant effect on the chelation reaction. As the temperature rises, both the chelation rate and the yield gradually increase, demonstrating that rising temperature contributes to the progress of the chelation process. When the temperature exceeds 85°C, the yield gradually decreases, but the chelation rate remains essentially unchanged, demonstrating that the reaction is inhibited when the temperature is too high. Analysis of variance shows that the yield of the 85°C group differs significantly from the 80°C group, and the difference from the other groups (other than 80°C) is extremely significant. The chelation rate at 85°C does not differ significantly from that at 80°C and 90°C, but the difference from the other groups (other than 80°C and 90°C) is extremely significant. Therefore, generally, the reaction temperature is controlled to be between 70 and 90°C, preferably between 80 and 85°C.

[0121] Experimental Example 2: Study on the effect of the mass ratio of peptide salts on chelation results. The pea oligopeptide aqueous solution was prepared at a concentration of 1% (i.e., 1 g / 100 mL), with a pH of 8.5 and a reaction temperature of 85°C. The reaction was carried out for 30 minutes under conditions of peptide salt mass ratios of 1:1, 2:1, 3:1, 4:1, and 5:1, respectively, and the effect of the peptide salt mass ratio on the yield and chelation rate was investigated.

[0122] As can be seen from Figure 28, as the mass ratio of peptide salt gradually increases, the chelation rate gradually decreases, but the yield gradually increases. The trends of the two indicators are inverse, and as the mass ratio of peptide salt increases, the mass of the salt decreases when the peptide concentration is kept constant. The decrease in salt mass is the main cause of the decrease in the chelation rate. However, when the mass ratio of peptide salt exceeds 3:1, the product yield also tends to decrease, but the decrease in the chelation rate gradually slows down. The condition in which the mass ratio of peptide salt is 3:1 is the condition in which the chelation yield reaches its highest value. Analysis of variance shows that the yield and chelation rate when the ratio is 3:1 differ significantly from other combinations, so generally, the mass ratio of pea oligopeptide to sodium selenite is controlled to be between 2 and 4:1.

[0123] Experimental Example 3: Effect of peptide concentration on yield and chelation rate The reaction was carried out for 30 minutes under the following conditions: a mass ratio of peptide salts of 3:1, a chelation pH of 8.5, and a chelation temperature of 85°C, with pea oligopeptide aqueous solutions at concentrations of 2% (i.e., 2g / 100mL), 3% (i.e., 3g / 100mL), 4% (i.e., 4g / 100mL), and 5% (i.e., 5g / 100mL). The effect of peptide concentration on yield and chelation rate was investigated.

[0124] As can be seen from Figure 29, with increasing concentration of the pea oligopeptide aqueous solution, both the chelation rate and yield show a trend of increasing first, becoming stable, and then decreasing further. Analysis of variance shows that the yield and chelation rate at a peptide concentration of 3% differ significantly from the two indicators in other sets (other than 4%), but the difference is not significant when compared to the two indicators at 4%. Therefore, the concentration of the pea oligopeptide aqueous solution is generally controlled to 3-5 g / 100 mL.

[0125] Experimental Example 4: Effect of reaction pH value on yield and chelation rate The pea oligopeptide aqueous solution was designed to a concentration of 4% (4g / 100mL), the peptide salt mass ratio was 3:1, the chelation temperature was set to 85°C, and the reaction was carried out for 30 minutes under conditions of chelation pH 6.5, 7, 8, 8.5, and 9, respectively. The effect of the reaction pH value on the yield and chelation rate was investigated.

[0126] As can be seen from Figure 30, the chelation rate and yield tend to increase overall with increasing solution pH, but once the pH exceeds 8, both indicators tend to decrease, and the rate of decrease gradually slows down. This proves that the chelation reaction yields better results in a weakly alkaline environment. Analysis of variance shows that at pH=8, the yield and chelation rate differ significantly from other combinations. Furthermore, the pH of the solution is approximately 8 when the pea oligopeptide and sodium selenite are completely dissolved.

[0127] Experimental Example 5: Effect of reaction time on yield and chelation rate The pea oligopeptide aqueous solution was prepared at a concentration of 4% (4g / 100mL), with a peptide salt mass ratio of 3:1. The chelation temperature was set to 85°C, and the chelation pH was 8.5. The reaction was carried out for 20, 30, 40, 50, and 60 minutes, and the effect of reaction time on yield and chelation rate was investigated.

[0128] As can be seen from Figure 31, the chelation rate and yield gradually increase as the chelation time is extended. When the chelation time exceeds 30 min, both the chelation rate and yield decrease and fluctuate stably within a certain range. Analysis of variance shows that the yield and chelation rate at 30 min differ significantly from the two indicators in the 20 min group, and also differ significantly from the other groups (groups other than the 20 min group). The two indicators between the 40, 50, and 60 min groups do not differ significantly.

[0129] Each of the above embodiments is solely for the purpose of illustrating the technical solutions of the present invention, and those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions to some or all of their technical features, and such modifications or substitutions will not depart from the essence of the corresponding technical solutions from the spirit of the technical solutions of each embodiment of the present invention.

Claims

1. A selen chelate pea oligopeptide, wherein the selen chelate pea oligopeptide contains a peptide fragment PPKIYP in a mass content of 25 ng / mg or more, and the selen chelate pea oligopeptide is A method using pepsin and performing hydrolysis for 4 hours under conditions of a pH of 2 and a temperature of 37°C. A method using trypsin and hydrolysis performed for 6 hours under conditions of a pH of 7.5 and a temperature of 37°C. After at least one of three digestion methods—one in which the temperature is kept constant at 37°C, hydrolysis is performed for 4 hours using pepsin under conditions where the pH value is 2, and then hydrolysis is performed for another 6 hours using trypsin under conditions where the pH value is 6.8—the selenium content after digestion does not change by more than 3% compared to the selenium content before digestion. The aforementioned selenium chelate pea oligopeptide has an acid-soluble protein content higher than 23%, a total nitrogen content higher than 23%, and components with a molecular weight of less than 1000u account for 85% or more, and a selenium content of 0.08 g / 100 g or more. The method for preparing the selen chelate pea oligopeptide includes reacting a mixed system of an aqueous solution of pea oligopeptide and sodium selenite at a temperature of 60 to 90°C for 20 minutes or more, and subjecting the resulting reaction product to alcohol precipitation and drying to obtain the selen chelate pea oligopeptide. The aforementioned mixed system has a mass ratio of pea oligopeptide to sodium selenite of 1 to 5:

1. The method for preparing the pea oligopeptide is as follows: A selenium chelate pea oligopeptide is characterized by mixing pea protein powder with water in a material-to-liquid ratio of 1:8 to 12, adjusting the pH of the material liquid to 8 to 10, controlling the temperature to 40 to 60°C, adding alkaline protease and neutral protease for enzymatic decomposition, with the dosage of both enzymes being 1.0 to 3.0% of the mass of the pea protein powder, the enzymatic decomposition time being 3 to 6 hours, the pea oligopeptide containing more than 80% of components with a molecular weight less than 1000u, and the mass concentration of the pea oligopeptide in the aqueous solution of the pea oligopeptide being 1 to 5 g / 100 mL.

2. The selenium chelate pea oligopeptide according to claim 1, characterized in that after the selenium chelate pea oligopeptide is heat-treated at 100°C or below for 2 hours, the selenium content is 97% or more of that before the heat treatment.

3. The selenium chelate pea oligopeptide according to claim 1 or 2, characterized in that after being subjected to acid-base treatment for 2 hours under conditions of pH 3 to 11 and temperature 37°C, the selenium content is 75% or more of that before the acid-base treatment.

4. A method for preparing selen chelate pea oligopeptide, comprising reacting an aqueous solution of pea oligopeptide with sodium selenite in a mixed system at 60 to 90°C for 20 minutes or more, and subjecting the resulting reaction product to alcohol precipitation and drying to obtain the selen chelate pea oligopeptide. The aforementioned mixed system has a mass ratio of pea oligopeptide to sodium selenite of 1 to 5:

1. The aforementioned selen chelate pea oligopeptide contains the peptide fragment PPKIYP in a mass content of 25 ng / mg or more. The aforementioned selenium chelate pea oligopeptide has an acid-soluble protein content higher than 23%, a total nitrogen content higher than 23%, and components with a molecular weight of less than 1000u account for 85% or more, and a selenium content of 0.08 g / 100 g or more. The method for preparing the pea oligopeptide is as follows: A method for preparing selenium chelate pea oligopeptide, characterized by mixing pea protein powder with water in a material-to-liquid ratio of 1:8 to 12, adjusting the pH of the material liquid to 8 to 10, controlling the temperature to 40 to 60°C, adding alkaline protease and neutral protease for enzymatic decomposition, administering the two enzymes in amounts of 1.0 to 3.0% of the mass of the pea protein powder, having an enzymatic decomposition time of 3 to 6 hours, the pea oligopeptide containing more than 80% of components with a molecular weight less than 1000u, and having an aqueous solution of the pea oligopeptide with a mass concentration of 1 to 5 g / 100 mL.

5. A method for using the selenium chelate pea oligopeptide described in any one of claims 1 to 3 in a healthcare food.

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