Brogia fusco-purpurea antioxidative peptide and preparation method thereof
Through alkaline extraction and enzymatic decomposition technology of red hairy algae, efficient antioxidant peptides were prepared, which solved the shortcomings in the research on algae peptides in aquatic plants, and achieved high purity and high antioxidant activity preparation of algae peptides, which were biologically active substances Research provides new directions.
Patent Information
- Application Number
- CN202411654383.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2025-01-03
AI Technical Summary
In the prior art, there are few researches on aquatic plant algae polypeptides, especially the development and utilization of antioxidant functions. Enzymatic methods are mainly used for animal-derived polypeptides, and the research and preparation methods of plant-derived polypeptides are relatively limited.
Using red hairy algae as material, ultrasonic-assisted protein extraction through alkaline method, enzymatic decomposition using papain, combined with ultrafiltration membrane and chromatography separation technology, a red hairy algae antioxidant peptide with excellent antioxidant function was prepared, including amino acid sequences. Peptides of YPCW, GYPYK, VLPYAK and NKLSSY.
A high-purity and high antioxidant activity of erythromycetes antioxidant peptide was obtained, which effectively prepared and applied algae peptides and provided new research and development directions for biologically active substances.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of marine algae biotechnology, and in particular to red hair algae antioxidant peptides and a preparation method thereof. Background Art
[0002] Bioactive peptides are a class of biologically active substances with important physiological functions that are derived from bacteria, fungi, animals and plants. Bioactive peptides not only have a variety of significant physiological functions, such as neuromodulation, hormone action, immune regulation, antithrombotic, antihypertensive, cholesterol-lowering, antibacterial, antiviral, anticancer, and antioxidant effects, but also have simple structures, strong stability, easy to be directly absorbed by the human body, and safe characteristics. Therefore, they have become a hot topic in the field of bioactive substance research in recent years.
[0003] At present, the main methods for preparing active peptides include enzymatic hydrolysis, microbial fermentation, and chemical synthesis. Enzymatic hydrolysis has become the most commonly used method for releasing bioactive peptides from proteins due to its mild production conditions, high safety, and the ability to obtain active peptides with specific functions. Studies on the preparation of bioactive peptides by enzymatic methods are mostly found in animal-derived peptides, while studies on plant-derived peptides are relatively rare. Wan Haixia et al. used pepsin to enzymatically hydrolyze the water-soluble and salt-soluble proteins of Polysiphonium, and the resulting peptides had strong DPPH free radical scavenging activity. Domestic and foreign research on the antioxidant activity of plant peptides is mostly limited to terrestrial plants, while there is little research on aquatic plants such as algae peptides. Algae live in the ocean, where living conditions are more complex, and may form protein structures and biological activities that are different from those of terrestrial plants. Therefore, algae peptides have more potential research value, and research on them is conducive to the in-depth development and utilization of algae and changing the production structure and application value of algae.
[0004] Bangia fusco-purpurea Lyngb., belonging to the Rhodophyta, Rhodophyta, and Rhodophyta, is a specialty of the southeast coast of China. It is rich in nutrients, with a protein content of up to 45%, which is conducive to the research and preparation of enzymatic peptides. Summary of the invention
[0005] The purpose of the present invention is to provide red hair algae antioxidant peptide and a preparation method thereof.
[0006] To achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a red hair algae antioxidant peptide, which is selected from any one of the following: a red hair algae antioxidant peptide with an amino acid sequence of YPCW, a red hair algae antioxidant peptide with an amino acid sequence of GYPYK, a red hair algae antioxidant peptide with an amino acid sequence of VLPYAK, and a red hair algae antioxidant peptide with an amino acid sequence of NKLSSY.
[0007] The second aspect of the present invention provides a method for preparing the above-mentioned red hair algae antioxidant peptide, comprising the following steps: S1: After cleaning and removing impurities from the red hair algae, drying at 60°C for 12 hours, crushing and passing through an 80-mesh sieve to obtain red hair algae powder; S2: Add pure water to the red hair algae powder, stir thoroughly to swell, adjust the pH to 9, perform ultrasonic-assisted extraction for 20 minutes, stir and extract at 50°C for 3 hours, centrifuge, discard the precipitate, take the supernatant, adjust the pH to 4.2, stand at 4°C for 12 hours, then centrifuge, discard the supernatant, take the precipitate and freeze-dry to obtain red hair algae protein; S3: dissolving the red hair algae protein with pure water, adding papain for enzymolysis, inactivating the enzyme, and obtaining a red hair algae protein hydrolysate; concentrating the red hair algae protein hydrolysate under reduced pressure at 60° C., adding anhydrous ethanol to stand for alcohol precipitation, centrifuging, discarding the precipitate, and taking the supernatant to obtain a red hair algae polypeptide liquid; S4: The red hair algae polypeptide liquid was separated and purified by an ultrafiltration membrane with a molecular weight cutoff of <2kD, and the components were separated and purified by DEAE-52 anion column and Sephadex LH-20 column in sequence, and the components with the highest antioxidant activity were collected; the red hair algae polypeptide liquid was separated and purified by an ultrafiltration membrane with a molecular weight cutoff of 2kD to 10kD, and the components were separated and purified by DEAE-52 anion column and Sephadex-G75 column in sequence, and the components with the highest antioxidant activity were collected; S5: determining the amino acid sequence of the peptides contained in the collected components with the highest antioxidant activity, and performing solid phase synthesis on the determined amino acid sequences to obtain red hair algae antioxidant peptides; In step S2, the mass volume ratio of the red algae powder to pure water is 1:25; In step S3, the mass volume ratio of the red hair algae protein to pure water is 1:20; the conditions for the enzymatic hydrolysis are: the amount of papain added is 3000U / g red hair algae protein, the enzymatic hydrolysis temperature is 50°C, the enzymatic hydrolysis pH is 6, and the enzymatic hydrolysis time is 2h; In step S4, the separation and purification conditions of the DEAE-52 anion column are: linear gradient elution with 0-2 mol / L NaCl solution at a flow rate of 3 mL / min; the separation and purification conditions of the Sephadex LH-20 column are: elution with deionized water at a flow rate of 3 mL / min; and the separation and purification conditions of the Sephadex-G75 column are: elution with deionized water at a flow rate of 3 mL / min.
[0008] The third aspect of the present invention provides the use of the above-mentioned red hair algae antioxidant peptide in the preparation of antioxidant functional products.
[0009] The advantages of the present invention are: (1) The present invention provides a red hair algae antioxidant peptide, wherein the red hair algae antioxidant peptide is selected from any one of the following: a red hair algae antioxidant peptide with an amino acid sequence of YPCW, a red hair algae antioxidant peptide with an amino acid sequence of GYPYK, a red hair algae antioxidant peptide with an amino acid sequence of VLPYAK, and a red hair algae antioxidant peptide with an amino acid sequence of NKLSSY. The results of the antioxidant activity assay show that the red hair algae antioxidant peptides YPCW, GYPYK, VLPYAK and NKLSSY provided by the present invention have excellent antioxidant functions.
[0010] (2) The present invention adopts alkaline method to extract red hair algae protein with ultrasound assistance, which has low salt content and high protein extraction rate. The isoelectric point method is used to separate proteins, and the obtained protein contains less small molecular compounds and has high purity. Papain is used to prepare red hair algae antioxidant peptides, and ultrafiltration membrane is used to prepare red hair algae polypeptides, which provides convenient conditions for the next step of chromatographic separation to obtain purer red hair algae antioxidant peptides. Different chromatographic separation methods are used according to the different molecular weights of red hair algae polypeptides, and the obtained red hair algae polypeptides have higher purity. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 : DEAE-52 anion column elution profile of BFPI components.
[0012] Figure 2 : DEAE-52 anion column elution profile of BFPII components.
[0013] Figure 3 : Sephadex LH-20 column elution profile of BFPI-1 fraction.
[0014] Figure 4 : Sephadex-G75 column elution profile of BFPII-3 fraction.
[0015] Figure 5 : Total ion current of BFPI-1-1 components.
[0016] Figure 6 : Total ion current of BFPII-3-1 component. DETAILED DESCRIPTION
[0017] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.
[0018] The antioxidant activity in the following examples was determined as follows: (1) Determination of DPPH free radical scavenging rate: DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) appears dark purple in ethanol and ethanol-water solution, and has maximum absorption at wavelengths of 517nm and 527nm. When DPPH free radical scavenger is present, its absorption gradually disappears due to pairing with its single electron, and its fading degree is stoichiometrically related to the number of paired electrons, so it can be quantitatively analyzed by spectrophotometry.
[0019] In the experimental group, 100 μL DPPH solution and 100 μL sample solution were added to the 96-well plate, and the plate was fully shaken and reacted in the dark for 30 minutes. The absorbance was measured at 517 nm (with 95% ethanol solution as zero). The blank group and the control group were replaced with an equal volume of 95% ethanol instead of DPPH solution and sample solution, respectively. Other operations were the same as those of the experimental group. The DPPH free radical scavenging rate was calculated according to the following formula.
[0020] Where: A o —Absorbance of control group; A i —Absorbance of sample group; A j —Absorbance of blank group.
[0021] (2) Determination of antioxidant capacity index (ORAC): The analysis was performed using a fluorescence microplate analyzer, using the azo compound AAPH (azobisisobutylamidine hydrochloride) as the source of peroxyl free radicals, FluoresceinSodium (fluorescein sodium) as the fluorescence indicator, and Trolox (water-soluble vitamin E) as the quantitative standard.
[0022] ORAC is the abbreviation of oxygen radical absorbance capacity, also known as the antioxidant capacity index. The free radicals in the ORAC analysis method mainly come from the hydrogen peroxide free radicals produced by the decomposition of the azo compound AAPH, and can also be hydroxyl free radicals produced during the Fenton reaction. Sodium fluorescein (FL) is used as a fluorescent probe to observe the decay process of the fluorescence intensity of the probe after the free radicals interact with the fluorescent probe. Trolox is used as an antioxidant standard substance to detect the ability of various antioxidants in the system to delay the decay of the probe fluorescence intensity, so as to evaluate the antioxidant capacity of the antioxidant.
[0023] FL was used as the oxidation substrate and AAPH as the free radical generator. FL was oxidized by free radicals and the fluorescence intensity gradually weakened. When an antioxidant was present, the fluorescence intensity weakened and was inhibited, and the antioxidant's ability to protect the FL fluorescence intensity was proportional to its concentration. The antioxidant Trolox was used as a standard to quantify the antioxidant capacity of the sample. 1. Based on the ORAC reaction, the reaction was carried out in a 75 mmol / L sodium phosphate buffer (pH=7.4) environment. The specific measurement operation was as follows: 50 μL of the sample to be tested and 50 μL of FL were added to each microwell of a 96-well plate, and after pre-setting at 37°C for 20 min, 100 μL of AAPH was quickly added to each well using a multichannel pipette to start the reaction (add samples according to the table below), and the microwell plate was placed in a fluorescence analyzer for continuous measurement at 37°C with an excitation wavelength of 485 nm and an emission wavelength of 538 nm. The fluorescence intensity of each well was measured every 2 min, and the measurement time was generally set until the fluorescence decay reached the baseline (about 120 min).
[0024] The ORAC experiment requires setting up two controls, namely the FL fluorescence natural decay control without the addition of free radicals (-AAPH) and the free radical action control in the absence of antioxidants (+AAPH). The antioxidant capacity of the sample is directly related to the delayed area of the fluorescence decay curve under the action of free radicals (NetAUC). The fluorescence intensity data of each microwell reaction obtained in the experiment is output to Excel through software for further processing. The absolute fluorescence intensity data of each microwell at different time points are compared with the blank fluorescence intensity of -AAPH and converted into relative fluorescence intensity f. The area under the fluorescence quenching curve (AUC) is calculated by the approximate integration method based on the relative fluorescence intensity. The area under the fluorescence decay curve can be approximately regarded as the sum of the areas of each trapezoid, and the formula is expressed as: AUC=0.5×(f0+f1)×Δt+0.5×(f1+f2)×Δt+…+0.5×(f x +f x+1 )×Δt+…+0.5×(f n-1 +f n )×Δt=0.5×[2×(f0+f1+…+f n-1 +f n )-f0-f n ]×Δt where f n represents the relative fluorescence intensity at the nth measurement point, and Δt is the time interval between two adjacent measurement points. Since Δt is set to 2 min in the ORAC determination method of this experiment, the formula can be simplified as: AUC = 2 × (f0 + f1 + ... + f n-1 +f n )-f0-f n The difference between the area under the fluorescence decay curve under the action of antioxidants and the area under the fluorescence decay curve under the action of free radicals in the absence of antioxidants, that is, the delayed part area of the fluorescence quenching curve NetAUC is the protection area of the antioxidant. The ORAC value of the oxygen free radical scavenging ability of the antioxidant, also known as the antioxidant capacity index of the antioxidant, is obtained by comparing the protection area of the fluorescence decay curve with the protection area of the standard antioxidant substance. The ORAC value is expressed in Trolox equivalents (μmolTrolox equivalents / μmol, μmolTrolox equivalents / ml and μmolTrolox equivalents / mg), and its calculation formula is: ORAC=[(AUC Sample -AUC +AAPH )÷(AUC Trolox -AUC +AAPH )]×(molarity ofTrolox / molarity ofsample) Example 1: S1: Alkaline isoelectric point method for protein extraction After the red algae was washed with pure water to remove impurities, it was dried at 60℃ for 12h and crushed through an 80-mesh sieve to obtain red algae powder. Weigh 50.00g of red algae powder, add pure water according to a mass volume ratio of 1:25 and stir thoroughly to swell, adjust the pH to 9, and extract at room temperature and ultrasonic power of 480w for 20min, and then stir and extract at 50℃ for 3h. After the extraction, centrifuge at 7000r / min for 15min, discard the precipitate, take the supernatant to adjust the pH to 4.2, stand at 4℃ for 12h, centrifuge at 7000r / min for 15min, discard the supernatant, take the precipitate and freeze-dry to obtain red algae protein.
[0025] S2: Enzymatic hydrolysis to prepare peptides Take the red hair algae protein, add pure water to dissolve it according to the mass volume ratio of 1:20, then add papain according to the enzyme substrate ratio [E] / [S] 3000U / g, enzymatically hydrolyze for 2h at a temperature of 50℃ and a pH of 6.0, and then inactivate the enzyme at 100℃ for 5min to obtain the red hair algae protein hydrolysate. The red hair algae protein hydrolysate is concentrated to 1 / 4 of the original volume under reduced pressure at 60℃, anhydrous ethanol is added to a final concentration of 70% by volume, and the mixture is allowed to stand for 2h and then centrifuged at 7000r / min for 20min, the precipitate is discarded, and the supernatant is taken to obtain the red hair algae polypeptide liquid.
[0026] S3: Ultrafiltration membrane purification of peptides The red hair algae polypeptide liquid was cut off with an ultrafiltration membrane with a molecular weight cutoff of 2kD to retain the components with a molecular weight <2kD, and the BFPI component was obtained; the red hair algae polypeptide liquid was cut off with ultrafiltration membranes with molecular weight cutoffs of 2kD and 10kD to retain the components with a molecular weight of 2kD to 10kD, and the BFPII component was obtained. The DPPH free radical scavenging rate and ORAC value of the BFPI and BFPII components were determined, and the results are shown in the table below.
[0027] Table 1 Antioxidant activity of BFPI and BFPII fractions S4: Peptide purification by anion column The BFPI component was passed through a DEAE-52 anion column (4×25 cm) and eluted with a linear gradient of 0-2 mol / L NaCl solution at a flow rate of 3 mL / min. The absorbance of the effluent sample was detected by a chromatogram acquisition analyzer HD-A. Figure 1 ) collected two peak fractions: BFPI-1 and BFPI-2; the BFPII fraction was passed through a DEAE-52 anion column (4×25 cm) and eluted with a linear gradient of 0-2 mol / L NaCl solution at a flow rate of 3 mL / min. The absorbance of the effluent sample was detected by a chromatogram acquisition analyzer HD-A. Figure 2 ) collected 3 peak components: BFPII-1, BFPII-2, and BFPII-3. The DPPH free radical scavenging rate and ORAC value of BFPI-1, BFPI-2, BFPII-1, BFPII-2, and BFPII-3 components were determined, and the results are shown in the table below. It can be seen from the table that the antioxidant activity of BFPI-1 and BFPII-3 components is significantly higher than that of the other three components, so BFPI-1 and BFPII-3 components were selected for further separation and purification.
[0028] Table 2 Antioxidant activity of BFPI-1, BFPI-2, BFPII-1, BFPII-2 and BFPII-3 fractions S5: Purification of peptides by dextran gel column The BFPI-1 fraction was passed through a Sephadex LH-20 column (2.5×60 cm) and eluted with deionized water at a flow rate of 3 mL / min and a detection wavelength of 210 nm. Figure 3 ) collected two peak fractions: BFPI-1-1 and BFPI-1-2; the BFPII-3 fraction was passed through a Sephadex-G75 column (2.5×60 cm) and eluted with deionized water at a flow rate of 3 mL / min and a detection wavelength of 210 nm. Figure 4) collected 2 peak components: BFPII-3-1 and BFPII-3-2. The DPPH free radical scavenging rate and ORAC value of BFPI-1-1, BFPI-1-2, BFPII-3-1 and BFPII-3-2 were determined, and the results are shown in the table below. It can be seen from the table that the antioxidant activity of BFPI-1-1 and BFPII-3-1 components is significantly higher than that of the other two components, so BFPI-1-1 and BFPII-3-1 components were subsequently selected for polypeptide composition and amino acid sequence analysis.
[0029] Table 3 Antioxidant activity of BFPI-1-1, BFPI-1-2, BFPII-3-1 and BFPII-3-2 fractions S6: Peptide composition and amino acid sequence analysis The amino acid sequences of the peptides contained in the BFPI-1-1 and BFPII-3-1 components were determined by liquid chromatography-mass spectrometry (LC-MS / MS).
[0030] 1) Pre-column: 300μm id×5mm, packed with Acclaim PepMap RPLC C18, 5μm, 2) Analytical column: 150μm id×150mm, packed with Acclaim PepMap RPLC C18, 1.9μm, 3) Mobile phase A: 0.1% formic acid, 2% ACN; 4) Mobile phase B: 0.1% formic acid, 80% ACN; 5) Flow rate: 600 nL / min; 6) Analysis time for each component: 120 min; From the total ion current ( Figure 5 , Figure 6 ) It can be seen that the BFPI-1-1 and BFPII-3-1 components are mixtures, and there is no main polypeptide with a significant abundance. From the molecular weight results, it can be seen that the molecular weight of the polypeptides in the sample is widely distributed, and the molecular weight range of the polypeptides with a higher content is 300-2000Da. The number of amino acids is estimated to be 3-20. Therefore, the next step of peptide de novo sequencing adopts a non-enzymatic method, and the peptide de novo sequencing uses Thermo's Obitrap FusionLumos mass spectrometer. The amino acid sequence information of the peptide is derived by manual interpretation of the secondary fragmentation fragments of the peptide.
[0031] The amino acid sequences of the top 4 peptides in the BFPI-1-1 fraction are FLPDLT, VLPYAK, FGEPAF, and KFLTN; the amino acid sequences of the top 6 peptides in the BFPII-3-1 fraction are YPCW, TTFTT, EEAP, NKLSSY, GYPYK, and EAAK. The amino acid sequences were sent to a professional biochemical company for Fmoc solid phase synthesis (purity>99%), and then the DPPH free radical scavenging rate, ABTS free radical scavenging rate, ·OH free radical scavenging rate, and ORAC value were determined. The sample concentrations for the DPPH free radical scavenging rate and ABTS free radical scavenging rate were 0.2000 mg / mL; the sample concentration for the OH free radical scavenging rate was 1.000 mg / mL. The results are shown in the table below. It can be seen from the table that the peptides VLPYAK, YPCW, NKLSSY, and GYPYK have good antioxidant activity.
[0032] Table 4 Free radical scavenging rate of 10 solid phase synthetic peptides Table 5. The half scavenging efficiency (IC) of 4 solid phase synthetic peptides on free radicals 50 ) and ORAC value Note: GSH stands for glutathione.
[0033] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. A red hair algae antioxidant peptide, characterized in that: The red hair algae antioxidant peptide is selected from any one of the following: a red hair algae antioxidant peptide with an amino acid sequence of YPCW, a red hair algae antioxidant peptide with an amino acid sequence of GYPYK, a red hair algae antioxidant peptide with an amino acid sequence of VLPYAK, and a red hair algae antioxidant peptide with an amino acid sequence of NKLSSY.
2. The method for preparing the antioxidant peptide of red hair algae as claimed in claim 1, characterized in that: The following steps are involved: S1: After cleaning and removing impurities from the red hair algae, drying at 60°C for 12 hours, crushing and passing through an 80-mesh sieve to obtain red hair algae powder; S2: Add pure water to the red hair algae powder, stir thoroughly to swell, adjust the pH to 9, perform ultrasonic-assisted extraction for 20 minutes, stir and extract at 50°C for 3 hours, centrifuge, discard the precipitate, take the supernatant, adjust the pH to 4.2, stand at 4°C for 12 hours, then centrifuge, discard the supernatant, take the precipitate and freeze-dry to obtain red hair algae protein; S3: dissolving the red hair algae protein with pure water, adding papain for enzymolysis, inactivating the enzyme, and obtaining a red hair algae protein hydrolysate; concentrating the red hair algae protein hydrolysate under reduced pressure at 60° C., adding anhydrous ethanol to stand for alcohol precipitation, centrifuging, discarding the precipitate, and taking the supernatant to obtain a red hair algae polypeptide liquid; S4: The red hair algae polypeptide liquid was separated and purified by an ultrafiltration membrane with a molecular weight cutoff of <2kD, and the components were separated and purified by DEAE-52 anion column and Sephadex LH-20 column in sequence, and the components with the highest antioxidant activity were collected; the red hair algae polypeptide liquid was separated and purified by an ultrafiltration membrane with a molecular weight cutoff of 2kD~10kD, and the components were separated and purified by DEAE-52 anion column and Sephadex-G75 column in sequence, and the components with the highest antioxidant activity were collected; S5: Determine the amino acid sequence of the peptides contained in the collected components with the highest antioxidant activity, perform solid phase synthesis on the determined amino acid sequences, and obtain red hair algae antioxidant peptides.
3. The preparation method according to claim 2, characterized in that: In step S2, the mass volume ratio of the red algae powder to pure water is 1:
25.
4. The preparation method according to claim 2, characterized in that: In step S3, the mass volume ratio of the red hair algae protein: pure water is 1:20; the conditions for the enzymatic hydrolysis are: the addition amount of papain is 3000U / g red hair algae protein, the enzymatic hydrolysis temperature is 50°C, the enzymatic hydrolysis pH is 6, and the enzymatic hydrolysis time is 2h.
5. The preparation method according to claim 2, characterized in that: In step S4, the separation and purification conditions of the DEAE-52 anion column are: linear gradient elution with 0~2 mol / L NaCl solution at a flow rate of 3 mL / min; the separation and purification conditions of the Sephadex LH-20 column are: elution with deionized water at a flow rate of 3 mL / min; the separation and purification conditions of the Sephadex-G75 column are: elution with deionized water at a flow rate of 3 mL / min.
6. Use of the red hair algae antioxidant peptide as claimed in claim 1 in the preparation of antioxidant functional products.