CN110776554B - Screening methods for antioxidant peptides - Google Patents
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Images
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- C—CHEMISTRY; METALLURGY
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- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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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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- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
本发明涉及一种抗氧化肽的筛选方法，包括如下步骤：米渣蛋白经过粉碎、脱脂、酶解、灭酶、离心、干燥处理后，得到酶解物；酶解物经溶解获得酶解液，向酶解液中添加金属有机框架材料MIL‑53(Cr)进行富集，得到混合液；向混合液中加入解吸剂，获得抗氧化肽提取物；其中，解吸剂包括ACN/H2O、ACN/H2O/H3PO4、ACN/H2O/TFA、ACN/H2O/CH3COOH、ACN/H2O/NH3·H2O中的至少一种。该筛选方法简单快速、选择性高；MIL‑53(Cr)与金属螯合肽间强烈的螯合作用对谷物蛋白中的金属螯合肽进行富集，再通过对富集金属螯合肽的MIL‑53进行解吸可以达到富集抗氧化肽的效果。
The invention relates to a screening method for antioxidant peptides, comprising the following steps: rice dregs protein is subjected to crushing, defatting, enzymolysis, enzyme killing, centrifugation and drying to obtain an enzymolysate; the enzymolysate is dissolved to obtain an enzymolysate solution , adding metal-organic framework material MIL-53(Cr) to the enzymatic hydrolysis solution for enrichment to obtain a mixed solution; adding a desorbent to the mixed solution to obtain an antioxidant peptide extract; wherein the desorbent includes ACN/H 2 O At least one of ACN/H 2 O/H 3 PO 4 , ACN/H 2 O/TFA, ACN/H 2 O/CH 3 COOH, ACN/H 2 O/NH 3 ·H 2 O. The screening method is simple, rapid and highly selective; the strong chelation between MIL-53(Cr) and metal chelate peptides enriches the metal chelate peptides in grain protein, and then through the enrichment of metal chelate peptides Desorption of MIL-53 can achieve the effect of enriching antioxidant peptides.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a screening method of antioxidant peptides.
Background
At present, the preparation methods of bioactive peptides (including antioxidant peptides) mainly include enzymolysis, synthesis, extraction, microbial fermentation, etc., wherein the preparation of bioactive peptides by enzymolysis has become the main method for industrial scale production. Since the biologically active peptides produced by the enzymatic method are a mixture, if the structure, activity and structure-activity relationship of the biologically active peptides produced by the enzymatic method are to be further studied, it is necessary to separate and purify the mixed peptides to determine the structure of the active peptides. The separation and purification methods are various, and the chromatographic separation technology, the membrane separation technology, the electrophoresis technology and the like and the combination of various methods are widely applied. The greatest disadvantage of these isolation and purification methods is that it is not possible to directly screen active peptides for a particular activity.
The raw materials for preparing the antioxidant peptide have wide sources, such as: the active peptide has antioxidant effect in food protein materials such as fermented milk, soybean, whey, corn, egg, fish, rapeseed, rice, etc. The rice yield of China is at the top of the world, the content of protein in the byproduct rice residue generated by producing starch sugar and extracting amino acid by using grains as raw materials is up to 50 percent (dry basis), but the protein is not effectively developed and utilized all the time. The existing research shows that the active peptide prepared by enzymolysis of cereal protein has stronger free radical scavenging capacity and the capacity of improving the immunocompetence of organisms, the cereal antioxidant active peptide is prepared by taking rice processing byproducts such as rice residue, broken rice, rice bran and the like as raw materials, an antioxidant is provided for people with oxidative stress and low immunity, and the active peptide has important social benefit and higher economic benefit. However, the current research on cereal antioxidant peptides focuses mainly on the simple examination of the antioxidant capacity of peptide mixtures after enzymolysis of cereal proteins, and little further relates to the separation, structural identification and antioxidant mechanism research of active antioxidant peptides. The reason is that the prior art has many separation steps of the active antioxidant peptide, and the separated index can not directly correspond to the active target, so that the target active peptide is difficult to be selectively separated, therefore, a novel method for enriching and purifying the active antioxidant peptide with simplicity, rapidness and high selectivity is researched, the enrichment performance and the recognition mechanism of the method on the target antioxidant peptide are determined, the change rule of the structure and the activity of the target antioxidant peptide in the enriching and purifying process is determined, the yield of the target active antioxidant peptide in the enriching and purifying process is further improved, and the method becomes a difficult problem to be urgently solved in the research of the antioxidant peptide at present.
Disclosure of Invention
Based on the above, the method for screening the antioxidant peptides is provided for solving the technical problems that the separation steps of the active antioxidant peptides are multiple, and the separated indexes cannot directly correspond to the active targets, so that the target active peptides are difficult to selectively separate.
A screening method of antioxidant peptides comprises the following steps:
s1, crushing the rice residue protein, degreasing, carrying out enzymolysis, inactivating enzyme, centrifuging and drying to obtain an enzymolysis product;
s2, dissolving the zymolyte to obtain an enzymolysis solution, adding metal organic framework material MIL-53(Cr) into the enzymolysis solution for enrichment, and centrifuging to obtain MIL-53(Cr) enriched with antioxidant peptide;
s3, adding a desorbent into the MIL-53(Cr) enriched with the antioxidant peptide in the step S2 to obtain the antioxidant peptide;
wherein the desorbent comprises ACN/H2O、ACN/H2O/H3PO4、ACN/H2O/TFA、ACN/H2O/CH3COOH、ACN/H2O/NH3·H2At least one of O.
In some embodiments, the degreasing comprises mixing the rice residue with petroleum ether, standing at room temperature, leaching for 2h, filtering with suction, drying, and repeating the steps for 3 times.
In some embodiments, the enzymolysis comprises the steps of adding 1mol/L NaOH and alkaline protease, and placing in a water bath at 60 ℃ for enzymolysis for 4 hours.
In some embodiments, the inactivating is performed at 90 ℃ for 15 min.
In some embodiments, the centrifugation is performed at 3500rpm for 15 min.
In some embodiments, the drying comprises vacuum drying, spray drying, or freeze drying.
In some embodiments, in the step of S2, the method further includes adjusting the pH of the mixed solution, where the pH is 3 to 7.
In some embodiments, in the step S2, the method further includes adding a salt to the mixed solution to adjust the ionic strength of the mixed solution, wherein the concentration of the salt is 0.1mol/L to 1mol/L, and the salt is NaCl.
In some embodiments, the temperature of the enrichment is 25 ℃ to 35 ℃, and the time of the enrichment is 2h to 6 h.
In the screening method, the zymolyte is metal chelating peptide, and the oxidation resistance is mainly realized by slowing down the generation of free radicals. The metal chelating peptide can chelate Fe in the organism3+And Cu2+So thatFree Fe3+And Cu2+Reduced concentration of Fe in equilibrium therewith2+And Cu+Is also reduced correspondingly, resulting in Fe2+And Cu+And the oxygen free radicals in cells generated by catalysis of transition metal ions are reduced, and the function of promoting the oxidation resistance of organisms is finally achieved. The MIL-53(Cr) adopted in the screening method contains active metal sites, can adsorb polypeptide, and has high adsorption selectivity. In addition, surprisingly, the desorbent added in the screening method can selectively screen the antioxidant peptide. Compared with the prior art, the invention also has the following beneficial effects: the screening method is simple and rapid, and has high selectivity; the strong chelation between the MIL-53(Cr) and the metal chelating peptide enriches the metal chelating peptide in the cereal protein, and then the MIL-53(Cr) enriched with the metal chelating peptide is desorbed to achieve the effect of enriching the antioxidant peptide.
Drawings
FIG. 1 is a synthesis scheme of metal organic framework MIL-53(Cr) and a working flow of the enrichment from rice residue protein enzymolysis liquid;
FIG. 2 is a Transmission Electron Micrograph (TEM) and a Scanning Electron Micrograph (SEM) of the metal-organic framework MIL-53 (Cr);
FIG. 3 is a fitted curve equation of antioxidant activity of antioxidant peptides;
FIG. 4 is a fitted curve equation of antioxidant activity of antioxidant peptides;
FIG. 5 is a high performance liquid chromatogram of rice residue zymolyte
FIG. 6 is a high performance liquid chromatogram of an eluate;
FIG. 7 is a mass spectrum and peptide sequence of antioxidant peptide.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1
FIG. 1 is a schematic flow chart of a screening method of antioxidant peptides. The screening method of the antioxidant peptide comprises the following steps:
s1, obtaining zymolyte after crushing, degreasing, enzymolysis, enzyme deactivation, centrifugation and drying of the rice residue protein, as shown in figure 5. The method specifically comprises the following steps:
pulverizing rice residue protein, sieving with 100 mesh sieve, extracting rice residue with petroleum ether at 80 deg.C in a ratio of 1:3(m/V) for degreasing, leaching at room temperature for 2 hr, filtering, drying at 60 deg.C, circulating for 3 times, and removing oil. Preparing a degreased rice residue solution with a substrate concentration of 7.5%, carrying out hydration dissolution for 0.5h at room temperature, heating the degreased rice residue solution to 60 ℃ in a constant-temperature water bath kettle, adjusting the pH value of the rice residue solution to 8.0 by using a 1mol/L NaOH solution, adding 1.25% Alcalase 2.4 alkaline protease, carrying out enzymolysis for 4h in a water bath at 60 ℃, inactivating enzyme for 15min at 90 ℃ after the enzymolysis is finished, carrying out centrifugal separation for 15min at 3500rpm after the hydrolysate is cooled to room temperature, and drying (vacuum drying, spray drying or freeze drying) to obtain an zymolyte. Wherein, the recovery rate of soluble nitrogen can reach 49.68% after the obtained zymolyte is subjected to freeze drying treatment.
S2, dissolving the zymolyte to obtain an enzymolysis solution, adding metal organic framework material MIL-53(Cr) into the enzymolysis solution for enrichment, and centrifuging to obtain MIL-53(Cr) enriched with the antioxidant peptide. The method specifically comprises the following steps:
preparing 100mL of 0.5mg/mL rice residue protease hydrolysate, weighing 50mg of MIL-53(Cr) and adding into the rice residue protease hydrolysate, and shaking up. Adjusting the pH value and the ionic strength of the solution to 6 and 1mol/L respectively, enriching the metal organic framework MIL-53(Cr) and the rice residue protein enzymatic hydrolysate for 4 hours at 25 ℃, and then centrifuging to obtain the MIL-53(Cr) enriched with antioxidant peptide, wherein the preparation method of the metal organic framework MIL-53(Cr) comprises the following steps:
accurately weighing 4g of chromium nitrate nonahydrate and 1.66g of terephthalic acid, dissolving in 50mL of deionized water, fully stirring for 3h, transferring all the liquid into a closed stainless steel reaction kettle with a polytetrafluoroethylene inner container (100mL), reacting in a 220 ℃ oven for 3d, washing the reaction product with absolute ethyl alcohol and deionized water for 3 times respectively, and placing at 60 ℃ for vacuum drying to obtain MIL-53 (Cr).
The prepared metal organic framework MIL-53(Cr) has the appearance shown in figure 2, and the particle size is 460 nm.
S3, adding a desorbent into the MIL-53(Cr) enriched in the antioxidant peptide in the step S2 to obtain an antioxidant peptide extract. The method specifically comprises the following steps:
collecting metal-organic framework material MIL-53(Cr) rich in a large amount of metal chelating peptide, and adding a desorbent to obtain antioxidant peptide extract. Wherein the desorbent comprises ACN H2O＝3:7(v/v)、ACN:H2O:H3PO4＝3:6:1(v/v)、ACN:H2O:TFA＝3:6:1(v/v)、ACN:H2O:CH3COOH 3:6:1(v/v) and ACN: H2O:NH3·H2O ═ 3:6:1 (v/v)).
S4 antioxidant activity and structure identification of antioxidant peptide
S41、IC50Refers to the mass concentration of the sample at which the clearance is 50%. Drawing curves according to the change of the clearance rate of samples with different mass concentrations and performing linear fitting on the curves to obtain IC50，IC50Lower indicates better antioxidant activity.
Accurately weighing 2.56mg of DPPH standard, and diluting to a constant volume of 100ml with anhydrous methanol to obtain a final concentration of 6.5 × 10-5mol/L. Ultrapure water was used to prepare 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL reduced glutathione solution and 0.4mg/mL, 0.8mg/mL, 1.2mg/mL, 1.6mg/mL, 2.0mg/mL rice residue protein hydrolysate. 0.5mL of desorbent with different concentrations, glutathione and rice residue protein zymolyte are taken to be mixed with 2.5mL of 6.5 multiplied by 10-5And (3) carrying out a reaction on a mol/L DPPH solution at room temperature in a dark place for 30min, measuring the absorbance Asample at 517nm, simultaneously measuring Acontrol by taking methanol as a blank as a control, wherein all the measured values are average values of three times, and the DPPH inhibition rate is calculated according to the formula of the clearance rate.
DPPH inhibition (%) (Acontrol-Asample)/Acontrol × 100
Referring to table 1 and fig. 3, a curve equation in which curves are plotted according to the change of the clearance rate of samples of different mass concentrations and linear fitting is performed on each curve and a parameter for eliminating DPPH radical activity.
TABLE 1
Preparing a pyrogallol solution with the concentration of 10mmol/L hydrochloric acid as a solvent and the concentration of 3mmol/L pyrogallol; preparing Tris-HCl buffer solution with the pH value of 8.2 and the concentration of 100 mmol/L. Placing the prepared solution in a water bath at 25 ℃ for heat preservation for 20min, taking 1mL of sample diluent, adding 0.25mL of pyrogallol, 2.25mL of Tris-HCl buffer solution and 1.5mL of distilled water, and oscillating and uniformly mixing; measuring the absorbance value at 325nm every 30s at constant temperature, reacting for 4.5min, and recording the absorbance value of the sample (A sample); using Tris-HCl buffer solution to replace a sample as a blank, and the rest operations are the same; the average rate of change per minute for the Δ blank and Δ samples was calculated.
Superoxide anion radical absorption (%) [ (Δ blank- Δ sample)/Δ blank ] x 100%
Referring to Table 2 and FIG. 4, for fitting curves and scavenging superoxide anion radical (O)2-·) The parameter (c) of (c).
TABLE 2
S42, further separating the eluent by high performance liquid chromatography, and separately collecting each elution peak at the wavelength of 220nm, wherein the high performance liquid chromatogram of the eluent is shown in figure 6;
s43, selecting ions to be tested through a primary mass spectrum, impacting parent ions of the ions to be tested into ordered series of ions through adjusting the energy of collision gas, and processing an ion fragment diagram in a mass spectrogram into a single-point charge bar diagram. Finally, the sequence information of the peptide fragment was analyzed by MaxEnt3 and pepseq software, as shown in fig. 7.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (9)
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