CN111996226A - Preparation method of soybean 7S antioxidant selenium peptide - Google Patents
Preparation method of soybean 7S antioxidant selenium peptide Download PDFInfo
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- CN111996226A CN111996226A CN202010756942.8A CN202010756942A CN111996226A CN 111996226 A CN111996226 A CN 111996226A CN 202010756942 A CN202010756942 A CN 202010756942A CN 111996226 A CN111996226 A CN 111996226A
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- 244000068988 Glycine max Species 0.000 title claims abstract description 71
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- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 32
- 239000003963 antioxidant agent Substances 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
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- JULROCUWKLNBSN-UHFFFAOYSA-N seleno-DL-cystine Natural products OC(=O)C(N)C[Se][Se]CC(N)C(O)=O JULROCUWKLNBSN-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A23L33/16—Inorganic salts, minerals or trace elements
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention discloses a preparation method of soybean 7S antioxidant selenium peptide, which comprises the following steps: (1) spraying selenium fertilizer on the leaf surfaces of the soybeans from the full-bloom stage of the soybeans, harvesting and drying the soybeans after the soybeans are ripe to obtain selenium-rich soybeans; (2) crushing selenium-rich soybeans and then degreasing the selenium-rich soybeans to obtain degreased soybean flour, and extracting 7S protein from the degreased soybean flour; (3) dissolving soybean 7S protein in water, adding protease into the protein solution for enzymolysis, inactivating, and centrifuging to obtain supernatant; (4) filtering the supernatant with hollow fiber ultrafiltration membrane, and freeze drying the filtrate to obtain the soybean 7S antioxidant selenium peptide. The soybean 7S antioxidant selenium peptide prepared by the invention has good antioxidant activity both inside and outside cells, and also has the advantages of high hydrolysis degree, high selenium content and the like.
Description
Technical Field
The invention relates to a preparation method of soybean 7S antioxidant selenium peptide.
Background
Soybean is an important vegetable protein source in human and animal husbandry, and the soybean protein mainly comprises four components of 2S, 7S, 11S and 15S, wherein the 7S protein accounts for about one third of the soybean protein and is rich in various components having important physiological functions on organisms, but the research on the soybean 11S protein is more at present, and the research on the soybean 7S protein is relatively less.
The trace element selenium is regarded as an important food-borne antioxidant. Its functions of scavenging oxygen free radicals and preventing the damage of biological membrane are widely accepted. In most organisms, the oxidoreductase functions to protect the organism from oxidative damage. However, selenium levels in food products are relatively low. According to the statistics published by the World Health Organization (WHO), there is a problem of selenium malnutrition in about 10 million people worldwide. Research shows that selenium deficiency can cause a series of diseases such as viral infection, keshan disease, reproductive dysfunction, thyroid dysfunction, mood disorder, cardiovascular diseases and the like. Therefore, proper selenium supplement can not only maintain the steady state of the organism, but also improve the oxidation resistance. Selenium is mostly present in polypeptide chains in the form of SeCys and SeMet, and the selenium peptide combines the dual effects of selenium and bioactive peptides.
Oxidative balance is an important state in the body. Reactive oxygen species are produced in the body due to endogenous reactions of the body (e.g., phagocytosis and respiration) and exposure of the body to physical and chemical substances (e.g., ultraviolet radiation and air pollutants). Excessive reactive oxygen species accumulation in the body can cause protein synthesis inhibition, DNA mutation, oxidized membrane phospholipid and Low Density Lipoprotein (LDL) modification, leading to a disturbance in cell homeostasis and even the development of severe diseases such as atherosclerosis, diabetes and cancer. In recent years, polypeptides having antioxidant activity prepared from food-derived proteins have been receiving extensive research attention due to their potential in developing new natural drugs and food ingredients. Compared with synthetic food antioxidants (such as BHA, BHT and the like), natural antioxidant peptides are safer and more reliable, and become a new trend for research of biology, medicine and food science.
Disclosure of Invention
The invention aims to provide a preparation method of soybean 7S antioxidant selenium peptide, aiming at solving the problems of less selenium-containing component, lack of antioxidant activity and the like in the prior art, the double-enzyme enzymolysis process provided by the invention can improve the release amount of peptide in the enzymolysis process and obviously enhance the antioxidant activity of the selenium peptide.
The technical solution of the invention is as follows:
a preparation method of soybean 7S antioxidant selenium peptide comprises the following steps:
(1) spraying selenium fertilizer on the leaf surfaces of the soybeans from the full-bloom stage, spraying the selenium fertilizer once every other day, harvesting the soybeans after the soybeans are ripe, and drying the soybeans in the sun to obtain selenium-enriched soybeans;
(2) pulverizing selenium-rich soybean, defatting to obtain defatted soybean powder, extracting 7S protein from defatted soybean powder, and freeze drying;
(3) dissolving soybean 7S protein in water, adding protease into the protein solution for enzymolysis, inactivating, and centrifuging to obtain supernatant;
(4) filtering the supernatant with hollow fiber ultrafiltration membrane, and freeze drying the filtrate to obtain the soybean 7S antioxidant selenium peptide.
Preferably, the selenium fertilizer contains 0.001-0.1% of sodium selenite.
Preferably, the 7S protein is extracted by the following method: mixing the defatted soybean powder with water of which the weight is 20-50 times that of the defatted soybean powder, adjusting the pH value to 8-11 by using NaOH solution, centrifuging after stirring, taking supernatant, adjusting the pH value of the supernatant to 6-7 by using HCl solution, centrifuging after stirring, taking the supernatant, finally adjusting the pH value of the supernatant to 3-5 by using HCl solution, centrifuging after stirring, and collecting precipitate to obtain 7S protein.
Further preferably, the extraction method of the 7S protein is as follows: mixing the defatted soybean powder with 30 times of water, adjusting the pH value to 9 by using NaOH solution, centrifuging after stirring, taking supernatant, adjusting the pH value of the supernatant to 6.4 by using HCl solution, centrifuging after stirring, taking the supernatant, finally adjusting the pH value of the supernatant to 4.8 by using HCl solution, centrifuging after stirring, and collecting precipitate to obtain 7S protein.
Preferably, the 7S protein is dissolved in water to make a 3-5% protein solution, most preferably 4%.
Preferably, the protease comprises neutral protease and alkaline protease, and the addition sequence is that the neutral protease is added for enzymolysis, and then the alkaline protease is added for enzymolysis.
More preferably, the addition amount of the neutral protease is 1500-2000U/g 7S protein, the addition amount of the alkaline protease is 5500-6500U/g 7S protein, most preferably, the addition amount of the neutral protease is 1800U/g 7S protein, and the addition amount of the alkaline protease is 6000U/g 7S protein.
Further preferably, the enzymolysis time is 1-3h, and the enzymolysis temperature is 50-60 ℃. Preferably, the enzymolysis time of the neutral protease is 2 hours, and the enzymolysis time of the alkaline protease is 2 hours.
Preferably, the hollow fiber ultrafiltration membrane has a molecular weight cut-off of 800-.
The invention has the beneficial effects that:
the soybean 7S antioxidant selenium peptide prepared by the invention has good antioxidant activity both inside and outside cells, and also has the advantages of high hydrolysis degree, high selenium content and the like. The selenium content in the selenium peptide product can be improved by 36 percent to the maximum as compared with soybean powder, the yield can reach 39.39 mu g/g, and the yield can reach the high level of 14.5g/100g soybean to the maximum. The selenium peptide prepared by the invention not only can be used as a safe selenium supplement to solve the problem of selenium deficiency of special people, but also can be used as a natural antioxidant to maintain the oxidation balance of the organism and prevent oxidation-related diseases, and simultaneously provides a new way for deep processing and recycling of natural soybeans.
Drawings
FIG. 1 shows the comparison of the enzymatic effects of five proteases.
FIG. 2 shows the effect of substrate concentration on the enzymatic effect.
FIG. 3 shows the effect of the amount of neutral protease added on the enzymatic hydrolysis.
FIG. 4 shows the effect of the digestion time of neutral protease on the digestion effect.
FIG. 5 shows the effect of the addition of alkaline protease on the enzymatic hydrolysis.
FIG. 6 shows the effect of the time of alkaline protease digestion on the digestion effect.
Fig. 7 is an evaluation of soybean 7S antioxidant peptides of different fractions.
Detailed Description
For better understanding of the present invention, the present invention is further described in detail with reference to the following examples, but those skilled in the art will appreciate that the following examples are not intended to limit the scope of the present invention, and that any changes and modifications based on the present invention are within the scope of the present invention.
The method for measuring the selenium content, the method for measuring the degree of hydrolysis, the method for measuring the DPPH-free radical clearance, and the intracellular antioxidant activity CAA EC will be briefly described50Method for measuring value.
(1) Determination of degree of hydrolysis:
determination of the degree of hydrolysis by the pH-STAT method
Calculating the formula:
wherein: DH: the degree of enzymatic hydrolysis of a protein is expressed as the percentage of peptide bonds in the starting protein that are cleaved; b: consumption of standard lye (mL); nb: the molar concentration (mol/L) of the standard alkali liquor; α: the dissociation degree of an α -amino acid, where pH is the pH of the reaction system and pK is the average dissociation constant of α -NH3+ released under the reaction conditions, typically pK ═ 7.0, [10(pH-pK) ]/[1+10(pH-pK) ]; mp is the total amount of substrate protein (g); htot peptide bonds (mmol/g) in the substrate protein, Htot is a constant for a particular protein, e.g., 7mmol/g soy protein.
(2) Measurement of DPPH.radical scavenging ability:
the antioxidant capacity of the selenium-rich soybean 7S antioxidant peptide is measured by a1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) method.
9.858mg of DPPH is dissolved by absolute ethyl alcohol, the volume is fixed to a 25mL brown volumetric flask, 1mmol/L DPPH mother liquor is prepared, and the mother liquor is refrigerated at 4 ℃ and placed for use. When in use, the mother liquor is diluted by 5 times by using absolute ethyl alcohol to prepare a solution of 0.2 mmol/L. 0.5mL of 0.2mmol/L of the absolute ethanol solution of DPPH was added with 0.5mL of distilled water (A0) and 0.5mL of the sample solution (A1), and 0.5mL of the sample solution (A2) was added with 0.5mL of the absolute ethanol solution, and absorbance values A0, A1 and A2 were measured at 517nm, respectively. The DPPH & free radical ratio of the sample was calculated by the following formula.
Dpph.radical clearance (%) [ 1- (a 1-a 2)/a0] x 100%
(3) Intracellular CAA EC50Determination of the value:
HepG-2 cells in logarithmic growth phase at 6X 104The number per well was seeded in 96-well plates (six in parallel) containing 100. mu.L of basal medium per well at 37 ℃ with 5% CO2After 24h, the medium was removed and rinsed once with 100. mu.L PBS. 100 μ L of a culture medium containing different concentrations of sample treatment solution (containing 25 μm DCFH-DA) was added to a 96-well plate (blank medium alone), and the sample concentration was 0,100,200,400,600,800,1200,1600,2000 μ g/L in this order. Culturing at 37 deg.C and 5% CO2 for 1 h. The 96-well plate was removed, the medium was removed, and the plate was rinsed once with 100. mu.L of PBS. Residual PBS and sample treatment were then removed and 100. mu.L HBSS (containing 600. mu.M ABAP) was added to the wells and detected with a fluorescent microplate reader at 37 ℃. The excitation wavelength is 485nm, and the emission wavelength is 538 nm.
EC50Represents the dosage required for the selenium-rich soybean 7S antioxidant peptide to cause 50% inhibition. The calculation was performed using SSPS 9.
(4) And (3) determination of selenium content:
hydride generation-fluorescence spectroscopy (HG-AFS) was used, see GB/T21729-2008.
Accurately weighing 0.5g of sample in an erlenmeyer flask, accurately weighing the sample to 0.0001g, adding 10mL of mixed acid solution prepared from concentrated nitric acid and perchloric acid according to the volume ratio of 9:1, sealing the erlenmeyer flask, and standing overnight. Placing the conical flask on an electric hot plate at 180 ℃ and heating until yellow smoke is exhausted and a small amount of white smoke is exhausted, adding 10mL of 6mol/L hydrochloric acid solution into the clear solution of 2mL, continuously heating until the solution is clear again, taking down the solution and fixing the volume to 25 mL. After the solution after constant volume is filtered, 5mL of each tube of 3 tubes are respectively put into a centrifuge tube, 0.4mL of concentrated hydrochloric acid and 0.2mL of potassium ferricyanide (100g/L) are added, and the mixture is kept stand for 6 hours. The 500 mu g/mL selenium single element standard solution is diluted to 5 mu g/mL, 10 mu g/mL, 20 mu g/mL, 40 mu g/mL, 60 mu g/mL and 100 mu g/mL, and then 10mL of each solution is added with 1mL of potassium ferricyanide solution and 2mL of concentrated hydrochloric acid. A standard blank is prepared by adding 2mL of potassium ferricyanide solution and 4mL of concentrated hydrochloric acid into 50mL of distilled water and standing for 6 h. The instrument conditions were as follows: the negative high voltage is 250V, the current of the selenium lamp is 50mA, the height of the atomizer is 8mm, the flow rate of carrier gas is 300mL/min, the flow rate of the shielding device is 800mL/min, a standard curve method is adopted, the reading time is 15s, the sample is automatically fed, and the injection amount is 0.5 mL. The selenium content of the sample was calculated as follows:
selenium content X ═ cV/1000m
Wherein X is the selenium content (mu g/g) of the sample; c is the selenium concentration (μ g/L) in the sample solution for measurement; v is sample liquid volume (mL); and m is the sample mass (g).
Example 1: screening test for hydrolase
1) Selenium-rich fertilizer (0.1g Na)2SeO3And 2.8g of sodium alginate dissolved in 1L of water) are sprayed on the leaf surfaces of the soybeans from the full-bloom stage, the fertilization period is four weeks, the fertilization is carried out once every two days, and the soybeans are harvested and dried for later use after being mature.
2) Pulverizing selenium-rich soybean, sieving with 200 mesh sieve, adding 30g soybean powder into 1000mL petroleum ether, stirring and leaching at room temperature for 2h, standing, removing petroleum ether after precipitation is completed, precipitating, and drying to obtain defatted soybean powder. Adding 1000mL of distilled water into 30g of defatted soybean powder, adjusting the pH value to 9.0 by using 1mol/mL of NaOH solution, stirring for 2 hours, centrifuging to obtain a supernatant, adjusting the pH value of the supernatant to 6.4 by using 1mol/mL of HCl solution, stirring for 30 minutes, centrifuging, collecting a precipitate to obtain 11S protein, adjusting the pH value of the supernatant to 4.8 by using 1mol/mL of HCl, stirring for 30 minutes, centrifuging to obtain 7S protein, freezing, drying and storing for later use.
3) Dissolving 3g of 7S protein in 100mL of distilled water, and performing enzymolysis with five common commercial enzymes including alkaline protease (Alcalase), neutral protease (Neutrase), compound protease (Protamex), Papain (Papain) and Trypsin (Trypsin) with the enzyme addition amount of 3% (w/w) and the enzymolysis time3h, the enzymolysis temperature is 50-60 ℃, the pH value of the enzymolysis liquid is respectively adjusted to the optimum pH value of each enzyme according to the commercial enzyme specification, after the enzymolysis is finished, the enzyme is inactivated by boiling water bath for 10min, and after cooling, the supernatant is centrifuged and freeze-dried for later use. Detecting the hydrolysis degree of the enzymolysis liquid, the selenium content of the enzymolysis product, DPPH clearance and CAA half maximum Effect Concentration (EC) of 50 percent of maximum effect50) The test results are shown in FIG. 1.
The results show that the degree of hydrolysis of alkaline protease is highest; the free radical clearance rate of the enzymolysis products of the neutral protease, the compound protease and the trypsin is highest but the three have no obvious difference; CAA EC of alkaline and neutral proteases50The lowest value; the selenium content of the products of alkaline and neutral proteases was higher but not significantly different compared to the other enzymes.
After comprehensive consideration, alkaline protease and neutral protein are selected as the best enzymes for the test, and a two-enzyme enzymolysis test is designed to determine the enzymolysis sequence.
Example 2: order of addition of protease
Dissolving 3g of 7S protein by using 100mL of distilled water, and then performing enzymolysis according to the sequence of neutral protease-alkaline protease and alkaline protease-neutral protease respectively, wherein the enzyme addition amount is 3% (w/w), the enzymolysis time is 3h, the enzymolysis temperature is 50-60 ℃, and the reaction is stopped by boiling water bath for 10 min. The degree of hydrolysis of the enzymatic hydrolysate, the selenium content of the enzymatic hydrolysate, DPPH.clearance and CAA EC were measured in the same manner as in example 150The test results are shown in Table 1.
Table 1: effect of the enzymolysis sequence on the enzymolysis Effect
| Neutral-basic | Basic-neutral | Neutral property | Basic property | |
| Selenium content (μ g/g) | 19.721±0.892a | 18.754±0.626a | 8.748±0.666b | 9.022±0.972b |
| Degree of hydrolysis (%) | 28.612±1.846a | 23.133±0.336b | 10.836±0.243c | 21.307±0.7b |
| DPPH.Rate of removal (%) | 35.951±3.44a | 28.711±2.073b | 29.209±1.859b | 22.603±2.416c |
| CAA EC50(μg/L) | 741.66±19.36a | 923.375±16.97b | 909.908±47.13b | 966.920±28.69b |
The results show that the hydrolysis degree, selenium content and antioxidant activity of the product after double-enzyme enzymolysis are obviously higher than those of single-enzyme enzymolysis. The hydrolysis effect of the neutral-alkaline protease is better than that of the alkaline-neutral protease, so that the enzymolysis sequence of the neutral protease and the alkaline protease is selected to carry out a single-factor experiment.
Example 3: optimization of enzymolysis conditions
1) Effect of substrate concentration on the Effect of enzymolysis
Adding 1-5 g of 7S protein into 100ml of distilled water, and then sequentially adding neutral protease and alkaline protease for enzymolysis, wherein the enzyme addition amount is 3% (w/w), the enzymolysis time is 3h, the enzymolysis temperature is 50-60 ℃, and the reaction is stopped by boiling water bath for 10 min. The degree of hydrolysis, DPPH.clear rate and CAAEC of the enzymatic hydrolysate were measured in accordance with the method of example 150The test results are shown in FIG. 2.
The results show that the DPPH-free radical clearance and CAA EC of the hydrolysate is achieved at a substrate concentration of 4%50The optimum value is reached. The hydrolysis effect becomes worse by continuing to increase the substrate concentration. Therefore, it is best to add 4g of 7S protein to 100ml of water, i.e., to prepare a 4% protein solution for enzymatic hydrolysis.
2) Influence of the addition of neutral protease on the enzymolysis effect
Adding 3g of 7S protein into 100ml of distilled water, and then sequentially adding neutral protease and alkaline protease for enzymolysis, wherein the enzymolysis conditions are as follows: adding neutral protease 1-5% (w/w), performing enzymolysis for 3h, stopping reaction in boiling water bath for 10min, cooling, adding alkaline protease 3% (w/w), performing enzymolysis for 3h, and stopping reaction in boiling water bath for 10 min. After cooling, centrifugation is carried out, and the supernatant is freeze-dried for later use. The degree of hydrolysis, DPPH.clearance and CAA EC of the enzymatic hydrolysate were measured in the same manner as in example 150The test results are shown in FIG. 3 below.
The results show that the DPPH free radical clearance of the enzymolysis products reaches the highest when the addition amount of neutral protease is 4 percent, while CAA EC50The optimum value was found when the amount of enzyme added was 3%. CAA EC taking into account enzymatic products50The difference between the values of the neutral protease addition amounts of 3% and 4% is small, and the neutral protease addition amount of 4% is finally selected, namely 1800U/g protein isThe best scheme is adopted.
3) Influence of enzymolysis time of neutral protease on enzymolysis effect
Selenium-enriched soy 7S protein was prepared as in example 1.
Adding 3g of 7S protein into 100ml of distilled water, and then sequentially adding neutral protease and alkaline protease for enzymolysis, wherein the enzymolysis conditions are as follows: adding neutral protease 3% (w/w), hydrolyzing for 1-5h, boiling water bath for 10min to terminate the reaction, cooling, adding alkaline protease 3% (w/w), and boiling water bath for 10min to terminate the reaction when the hydrolysis time is 3 h. After cooling, centrifugation is carried out, and the supernatant is freeze-dried for later use. The degree of hydrolysis, DPPH.clearance and CAA EC of the enzymatic hydrolysate were measured in the same manner as in example 150The test results are shown in FIG. 4 below.
The results show the degree of hydrolysis of the enzymatic hydrolysate and the CAA EC of the enzymatic hydrolysate50The values are smaller when neutral protease is hydrolyzed for 2h and 3h, and the DPPH & free radical clearance of the enzymolysis products reaches the maximum value when neutral protease is hydrolyzed for 2h, so that the enzymolysis time of the neutral protease is 2h, which is the best scheme.
4) Influence of alkaline protease addition on enzymolysis effect
Adding 3g of 7S protein into 100ml of distilled water, and then sequentially adding neutral protease and alkaline protease for enzymolysis, wherein the enzymolysis conditions are as follows: adding neutral protease in an amount of 3% (w/w), hydrolyzing for 3h, stopping the reaction in boiling water bath for 10min, cooling, adding 1-5% (w/w) alkaline protease, performing enzymolysis for 3h, and stopping the reaction in boiling water bath for 10 min. After cooling, centrifugation is carried out, and the supernatant is freeze-dried for later use. The degree of hydrolysis, DPPH.clear rate and CAAEC of the enzymatic hydrolysate were measured in accordance with the method of example 150The test results are shown in FIG. 5.
The results show that the hydrolysis degree and DPPH free radical clearance of the enzymolysis products are smaller when the addition amount of the alkaline protease is 3 percent and 4 percent, and the difference between the CAA EC and the DPPH free radical clearance is smaller50The value is the lowest when the enzyme is added in 3 percent, and finally the optimum scheme is determined that the addition amount of the alkaline protease is 3 percent, namely 6000U/g protein.
5) Influence of hydrolysis time of alkaline protease on enzymolysis effect
Adding 3g of 7S protein into 100ml of distilled water, and sequentially addingCarrying out enzymolysis by using sexual protease and alkaline protease under the enzymolysis conditions: adding neutral protease in an amount of 3% (w/w), performing enzymolysis for 3h, stopping the reaction in boiling water bath for 10min, cooling, adding alkaline protease in an amount of 3% (w/w), performing hydrolysis for 1-5h, and stopping the reaction in boiling water bath for 10 min. After cooling, centrifugation is carried out, and the supernatant is freeze-dried for later use. The degree of hydrolysis, DPPH.clearance and CAA EC of the enzymatic hydrolysate were measured in the same manner as in example 150The test results are shown in FIG. 6 below.
The results show that the hydrolysis degree of the enzymolysis liquid and the enzymolysis product CAA EC50The difference between the values of the alkaline protease and the alkaline protease is small when the alkaline protease is subjected to enzymolysis for 2 hours and 3 hours, and the DPPH-free radical clearance of the enzymolysis products is the maximum when the alkaline protease is subjected to enzymolysis for 2 hours, so that the optimal scheme is that the enzymolysis time of the alkaline protease is 2 hours.
Example 4: selenium peptide comparison of different fractions
Adding 4g of 7S protein into 100ml of distilled water, and then sequentially adding neutral protease and alkaline protease for enzymolysis, wherein the enzymolysis conditions are as follows: adding neutral protease 4% (w/w), performing enzymolysis for 2h, stopping the reaction in boiling water bath for 10min, cooling, adding alkaline protease 3% (w/w), performing enzymolysis for 2h, and stopping the reaction in boiling water bath for 10 min. Cooling, centrifuging, collecting supernatant, performing fractional classification with ultrafiltration membranes with different molecular weight cut-off, coarse-filtering with 6000 Dalton hollow fiber membrane before classification, and lyophilizing the obtained sample. Detecting the selenium content, DPPH-clearance and CAA EC in the product50The test results are shown in FIG. 7 below.
The results show that the selenium content in the selenium peptides with different molecular weights has no obvious difference, but the DPPH-free radical clearance rate and CAA EC of the selenium peptide with the molecular weight less than 1000 daltons50Values are given for other molecular weights of the selenium peptide. Therefore, the hollow fiber ultrafiltration membrane with the molecular weight cutoff of 1000 daltons is selected to be optimal, the DPPH & free radical clearance of the final product reaches 35.27%, and CAA EC50It was 320.05. mu.g/L.
The product prepared by the invention has obviously improved selenium content, hydrolysis degree, chemical and intracellular antioxidant activity, and can be used as a good dietary supplement or antioxidant related additive to be applied to food, health care products and medicines. Meanwhile, the selenium content in the selenium peptide product is improved by 36 percent compared with soybean powder, the selenium peptide product reaches 39.39 mu g/g, and the yield can reach the high level of 14.5g/100g soybean.
Claims (9)
1. A preparation method of soybean 7S antioxidant selenium peptide is characterized by comprising the following steps:
(1) spraying selenium fertilizer on the leaf surfaces of the soybeans from the full-bloom stage, spraying the selenium fertilizer once every other day, harvesting the soybeans after the soybeans are ripe, and drying the soybeans in the sun to obtain selenium-enriched soybeans;
(2) pulverizing selenium-rich soybean, defatting to obtain defatted soybean powder, extracting 7S protein from defatted soybean powder, and freeze drying;
(3) dissolving soybean 7S protein in water, adding protease into the protein solution for enzymolysis, inactivating, and centrifuging to obtain supernatant;
(4) filtering the supernatant with hollow fiber ultrafiltration membrane, and freeze drying the filtrate to obtain the soybean 7S antioxidant selenium peptide.
2. The method of preparing soybean 7S antioxidant selenium peptide as claimed in claim 1, wherein: the selenium fertilizer contains 0.001-0.1% of sodium selenite.
3. The method of preparing soybean 7S antioxidant selenium peptide as claimed in claim 1, wherein: the extraction method of the 7S protein comprises the steps of mixing defatted soybean powder with water of which the weight is 20-50 times that of the defatted soybean powder, adjusting the pH value to 8-11 by using NaOH solution, stirring, centrifuging to obtain supernatant, adjusting the pH value of the supernatant to 6-7 by using HCl solution, stirring, centrifuging to obtain supernatant, adjusting the pH value of the supernatant to 3-5 by using HCl solution, stirring, centrifuging, and collecting precipitate to obtain the 7S protein.
4. The method of preparing soybean 7S antioxidant selenium peptide as claimed in claim 3, wherein: the extraction method of the 7S protein comprises the steps of mixing the defatted soybean powder with 30 times of water by weight, adjusting the pH value to 9 by using NaOH solution, centrifuging and taking supernatant after stirring, adjusting the pH value of the supernatant to 6.4 by using HCl solution, centrifuging and taking the supernatant after stirring, adjusting the pH value of the supernatant to 4.8 by using HCl solution, centrifuging and collecting precipitate after stirring to obtain the 7S protein.
5. The method of preparing soybean 7S antioxidant selenium peptide as claimed in claim 1, wherein: dissolving 7S protein in water to obtain 3-5% protein solution.
6. The method of preparing soybean 7S antioxidant selenium peptide as claimed in claim 1, wherein: the protease comprises neutral protease and alkaline protease, and the adding sequence is that the neutral protease is firstly added for enzymolysis, and then the alkaline protease is added for enzymolysis.
7. The method of claim 6, wherein the soybean 7S antioxidant selenium peptide comprises: the addition amount of the neutral protease is 1500-2000U/g 7S protein, and the addition amount of the alkaline protease is 5500-6500U/g 7S protein.
8. The method of claim 6, wherein the soybean 7S antioxidant selenium peptide comprises: the enzymolysis time is 1-3h, and the enzymolysis temperature is 50-60 ℃.
9. The method of preparing soybean 7S antioxidant selenium peptide as claimed in claim 1, wherein: the molecular weight cut-off of the hollow fiber ultrafiltration membrane is 800-.
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