CN114774499A - Extraction method and application of pearl oil apricot antioxidant peptide APHs-1-c - Google Patents
Extraction method and application of pearl oil apricot antioxidant peptide APHs-1-c Download PDFInfo
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- 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
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
- A23J1/146—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds by using wave energy or electric current
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- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
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- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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Abstract
The invention belongs to the technical field of polypeptide extraction, and particularly relates to an extraction method and application of pearl oil apricot antioxidant peptide APHs-1-c. The extraction method is realized by the following steps: (1) extracting almond protein; (2) carrying out enzymolysis to obtain APHs; (3) carrying out tangential cross flow membrane separation on the APHs to obtain three components, collecting the three components, freeze-drying the three components, and respectively recording the three components as APHs-1, APHs-2 and APHs-3; (4) and (3) separating the APHs-1 by using Sephadex G-25 as column chromatography packing, and collecting a final target substance APHs-1-c. The almond protein hydrolysate APHs-1-c obtained by separation has obvious protective effect on oxidative damage of HepG2 cells caused by TBHP, and has very strong scavenging effect on intracellular ROS. APHs-1-c can also protect HepG2 cells from oxidative stress damage caused by TBHP by activating the Keap1-Nrf2/ARE pathway.
Description
Technical Field
The invention belongs to the technical field of polypeptide extraction, and particularly relates to an extraction method and application of pearl oil apricot antioxidant peptides APHs-1-c.
Background
Apricot (A), (B)Prunus armeniaca L.) Is prepared from Rosaceae (Rosaceae) Larresiaceae (Larrea fruticosa (L.))Prunoideae) Apricot genus plant (Armeniaca Mil.l). The pearl oil apricot is a variation variety of apricot of Rosaceae, and is found in Xintai city of Shandong province in 1956. The pearl oil apricot has stable character, strong stress resistance, good fruit quality and high commodity value. In 2008, a large area of stone box villages in ancient city of Jinan, Shandong province are planted to form a thousand mu pearl oil apricot base, so that the stone box pearl apricot is also called as stone box pearl apricot.
The variety of functional foods in China is wide, so that the selection of consumers is diversified. Among various functional foods, bioactive peptides have the advantages of easy digestion, high safety, obvious efficacy, rich sources and the like, and thus become a research hotspot in the field of functional health foods. Biologically Active Peptides (BAPs) are peptides consisting of 2 to 20 amino acid residues. The bioactive peptide has multiple activities, such as antioxidant, antiinflammatory, antibacterial, blood pressure lowering, and blood lipid lowering.
At present, few reports are reported on further studies on extraction of almond protein.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for extracting antioxidant peptides APHs-1-c of pearl oil apricot.
The invention also aims to provide application of the pearl oil apricot antioxidant peptide APHs-1-c in antioxidant active products.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides a method for extracting antioxidant peptide APHs-1-c of pearl oil apricot, which comprises the following steps:
(1) weighing defatted almond powder, placing into a beaker, adding deionized water, adjusting pH to 8, performing ultrasonic extraction, centrifuging the extract, and leaving supernatant; adjusting the pH value of the obtained supernatant to 4.5, standing, centrifuging again, collecting precipitate, washing with water, and freeze-drying the precipitate in a vacuum freeze-drying machine to obtain almond protein;
(2) weighing almond protein, dissolving in deionized water to prepare a protein solution, preheating in a water bath, adding protease, and carrying out enzymolysis while keeping the pH value of the protein solution unchanged; after the enzymolysis is finished, stopping the reaction, then centrifuging, taking supernate, and carrying out vacuum freeze drying to obtain almond polypeptide powder, which is marked as APHs;
(3) sequentially separating the APHs by a tangential cross flow membrane with the molecular weight cutoff of 3 kDa and 10 kDa to obtain three components, collecting the three components, freeze-drying, and respectively recording the three components as APHs-1, APHs-2 and APHs-3;
(4) separating APHs-1 by using Sephadex G-25 as column chromatography filler, and naming APHs-1-a, APHs-1-b and APHs-1-c according to the peak appearance sequence at the wavelength of 220 nm; eluting for multiple times, respectively collecting eluates of the three components, vacuum freeze drying, and collecting final target APHs-1-c.
Further, in the step (1), the liquid-material ratio of the degreased almond powder to the deionized water is 25-35: 1; the ultrasonic extraction conditions are as follows: the power is 200-; the centrifugation is performed for 20 min under the condition of 7000 r/min; the re-centrifugation is carried out for 15min under the condition of 7000 r/min.
Further, in the step (2), the mass fraction of the protein solution is 2%; preheating the water bath to 70 ℃; the protease is one or more of neutral protease, compound protease, alkaline protease, flavourzyme and papain; the addition amount of the enzyme is 4-6%; the temperature of the enzymolysis is 45-55 ℃, the pH value is 6.0-10.0, and the enzymolysis time is 6 h; the termination reaction is heating for 15min at 100 ℃; the centrifugation is carried out for 20 min under the condition of 7000 r/min.
Further, in the step (3), the molecular weights of the APHs-1, the APHs-2 and the APHs-3 are respectively less than 3 kDa, 3 kDa-10 kDa and more than 10 kDa
The invention also provides application of the pearl oil apricot antioxidant peptide APHs-1-c in preparing antioxidant products.
The invention has the beneficial effects that:
(1) the method adopts an ultrasonic-assisted method to extract the almond protein, improves the protein extraction rate, and achieves (61.57 +/-0.364)%, under the synergistic effect of all steps;
(2) the almond protein extracted by the invention has excellent physical and chemical properties such as solubility, water retention, oil retention, foamability, foam stability, emulsibility, emulsion stability and the like, and can be used as a food additive.
(3) The almond protein hydrolysate APHs-1-c obtained by separation has obvious protective effect on oxidative damage of HepG2 cells caused by TBHP, and has very strong scavenging effect on intracellular ROS. APHs-1-c can also protect HepG2 cells from oxidative stress damage caused by TBHP by activating the Keap1-Nrf2/ARE pathway.
Drawings
FIG. 1 is a graph showing the effect of liquid-to-liquid ratio on the extraction rate of almond protein in example 1.
FIG. 2 is a graph showing the effect of temperature on the extraction rate of almond protein in example 1.
FIG. 3 is a graph showing the effect of time on the extraction rate of almond protein in example 1.
FIG. 4 is a graph showing the effect of ultrasonic power on the extraction rate of almond protein in example 1.
FIG. 5 is a graph showing the effect of different proteolytic enzymes on the degree of proteolysis of almond in example 2.
FIG. 6 is a graph showing the effect of different proteolytic cleavages on DPPH radical clearance of almond protein hydrolysate in example 2.
FIG. 7 is a graph showing the effect of enzyme dosage on the degree of proteolysis and DPPH radical scavenging capacity in example 2.
FIG. 8 is a graph showing the effect of enzyme digestion time on the degree of proteolysis and DPPH radical scavenging ability in example 2.
FIG. 9 is a graph of ABTS radical clearance for each fraction after ultrafiltration in example 3.
FIG. 10 is a diagram showing the reduction kinetics of iron ions in each fraction after ultrafiltration in example 3.
FIG. 11 is the elution profile of APHs-1 in example 3.
FIG. 12 is a graph of the effect of APHs-1-c on the survival of oxidatively damaged HepG2 cells in example 3.
FIG. 13 is a graph of the effect of APHs-1-c on ROS scavenging ability in HepG2 cells in example 3.
FIG. 14 is a graph showing the effect of APHs-1-c on the expression of Keap1, Nrf2 and HO-1 in HeapG2 cells in example 3.
FIG. 15 is a diagram of the peaks of the APHs-1-c mass spectrum isolated in example 4.
FIG. 16 is a schematic diagram showing that APHs-1-c can protect HepG2 cells from oxidative damage by regulating the Keap1-Nrf2/ARE signaling pathway.
Detailed Description
The technical solution of the present invention is further explained and illustrated by the following specific examples.
The almond powder used by the invention is degreased almond powder of pearl oil apricots, and the almond variety is pearl oil apricots, which are identified by Ninglin research institute of fruit trees of academy of agricultural sciences in Shandong province. Pulverizing semen Armeniacae amarum, sieving with 40 mesh sieve, and extracting semen Armeniacae amarum powder with n-hexane under stirring at room temperature. Air drying the defatted semen Armeniacae amarum powder in a fume hood, and storing at-20 deg.C.
The basic components are shown in table 1.
TABLE 1
Example 1 extraction of Pearl oil apricot Kernel protein
Weighing 100 g of degreased almond powder, placing the degreased almond powder in a beaker, adding 300g of deionized water, adjusting the pH value by using 1 mol/L NaOH solution, and setting ultrasonic power, temperature and ultrasonic time; centrifuging the extractive solution at 7000 r/min for 20 min, and collecting supernatant. Adjusting the pH value of the obtained supernatant to 4.5 by using 1 mol/L HCL, standing for 30min, centrifuging at 7000 r/min for 15min, collecting the precipitate, washing with water for 3 times, and freeze-drying the precipitate in a vacuum freeze-drying machine to obtain the almond protein, and storing at-20 ℃ for later use. The purity of the almond protein powder is 86.5 percent by Kjeldahl method detection.
As the liquid-material ratio increases, the ultrasonic conditions are as follows: extracting at 45 deg.C for 30min at 250 w; the extraction rate of the almond protein shows a trend of increasing firstly and then smoothing, and as can be seen from figure 1, when the liquid-material ratio is 30:1, the extraction rate of the almond protein reaches the maximum (53.84%), and the extraction rate of the protein tends to smooth along with the further increase of the liquid-material ratio, which shows that the dissolution amount of the almond protein reaches the maximum under the condition. In addition, with the further increase of the feed-liquid ratio, the components of some non-protein substances are gradually increased, and the reaction system is gradually enlarged, so that the subsequent precipitation and collection of protein are not facilitated.
With the increase of the temperature (other conditions are the same as above), the extraction rate of the almond protein shows a trend of increasing firstly and then decreasing. As can be seen from FIG. 2, the extraction rate of almond protein reached a maximum of 55.17% at a temperature of 45 ℃. When the temperature is higher than 45 ℃, the extraction rate of the almond protein is obviously reduced, and the protein is possibly difficult to dissolve out due to irreversible chemical changes such as denaturation and the like of the protein caused by overhigh temperature.
With the increase of the extraction time (other conditions are the same as above), the extraction rate of the almond protein gradually increases. As shown in figure 3, the extraction rate of the almond protein is rapidly improved with the increase of time between 10 min and 30 min. After 30min, the protein extraction rate is still improved, but is nearly flat, because the almond powder gradually swells fully and the protein is dissolved out to reach saturation with the increase of time.
With the increase of the ultrasonic power (other conditions are the same as above), the extraction rate of the almond protein is increased and then decreased. As shown in FIG. 4, when the ultrasonic power reaches 250W, the extraction rate of the almond protein reaches the maximum value. The extraction rate of the almond protein is gradually reduced by continuously increasing the ultrasonic power, probably because the stronger ultrasonic power generates overhigh thermal effect, so that partial protein is denatured and difficult to dissolve.
Example 2 enzymolysis Process of Almond protein
Weighing a certain amount of almond protein, dissolving the almond protein in deionized water to prepare a protein solution with the mass fraction of 2%, preheating for 15min in 70 ℃ water bath, then adding a proper amount of protease, carrying out enzymolysis for a certain time at the enzymolysis temperature of 55 ℃ and under the condition that the pH is =8, continuously dropwise adding 1 mol/L NaOH to keep the pH of the protein solution unchanged in the process, and recording the consumption of the NaOH solution for calculating the hydrolysis degree of the almond protein. Heating at 100 deg.C for 15min to stop reaction after enzymolysis, centrifuging at 7000 r/min for 20 min to obtain supernatant, vacuum freeze drying to obtain semen Armeniacae amarum polypeptide powder, and recording as APHs, and storing at-20 deg.C for use.
The almond protein is enzymolyzed by 5 proteases for 2.5h, the enzyme addition is 6%, and the curve of the change of the hydrolysis degree along with the time is shown in figure 16. The 5 protease hydrolysis reactions are in accordance with a typical enzymatic process curve, and within the first 1 h, the 5 protease hydrolysis process of the almond protein is relatively rapid. After 1 hour, only the alkaline protease still has a relatively obvious promoting effect on the hydrolysis degree of the almond protein, and the effects of the other 4 proteases gradually become slow.
The DPPH free radical scavenging rate of the almond proteolysis product generated by 5 protease zymohydrolysis is shown in figure 6. The antioxidant capacity of the polypeptide obtained by enzymolysis of 5 proteases is different, wherein the antioxidant capacity of the enzymolysis product of the alkaline protease is the strongest, and the DPPH free radical clearance rate reaches 49.87%. 5, the protease enzymolysis product has the following strength and weakness relationship for clearing DPPH free radicals: alkaline protease, papain, compound protease, flavourzyme and neutral protease.
As shown in figure 7, alkaline protease is selected for enzymolysis, the enzymolysis time is 2.5h, the enzyme adding amount is 6%, the hydrolysis degree of the reaction system is gradually increased along with the increase of the enzyme using amount, the combination probability of protein and enzyme is increased along with the increase of the enzyme using amount, and the hydrolysis degree is promoted to be gradually increased. The DPPH free radical scavenging activity of the enzymolysis products shows a rising trend along with the increase of the enzyme dosage, and reaches a maximum value (48.67%) when the enzyme dosage is 5%.
As shown in FIG. 8, alkaline protease is selected for enzymolysis for 2.5h, the enzyme addition amount is 6%, and the hydrolysis degree of protein gradually increases with the increase of enzymolysis time. The scavenging capacity of the enzymolysis products to DPPH free radicals is in a trend of increasing firstly and then decreasing, and the clearance rate reaches the maximum value of 47.86% at 2.5 h.
EXAMPLE 3 Ultrafiltration separation of APHs
(1) Sequentially separating the APHs through a tangential cross-flow membrane with the molecular weight cutoff of 3 and 10 to obtain three components with the molecular weights of 3 kDa, 3 kDa-10 kDa and >10 kDa. Collecting three components of <3 kDa, 3 kDa-10 kDa and >10 kDa, freeze-drying, and recording as APHs-1, APHs-2 and APHs-3, and storing at-20 ℃ for later use. And measuring the in vitro antioxidant capacity of the ultrafiltration components for comparison.
(2) The ABTS free radical clearance of each component after ultrafiltration is shown in FIG. 9. As can be seen from FIG. 9, each group showed strong ABTS free radical scavenging ability in the concentration range of 0.25-4.00 mg/mL, and was dose-dependent. Wherein APHs-1 exhibits strong ABTS free radical scavenging activity at all concentrations, and IC thereof500.42 mg/mL was reached.
(3) Reducing power of iron ions of each component after ultrafiltration
As can be seen from FIG. 10, at 0.25-4.00 mg/mL, the absorbance of each component polypeptide at 593 nm at low concentration is low, which indicates that the iron ion reducing capability is low, and the absorbance gradually increases with the increase of the concentration, which indicates that the iron reducing capability is gradually increased. Wherein the iron ion reducing capability of APHs-1 in each group is strongest, and the absorbance at 593 nm can reach 0.53 when the concentration is 4.00 mg/mL, which is far greater than the absorbance at 593 nm of APHs, which is 0.193.
(4) Analysis of amino acid composition of each fraction after ultrafiltration
The amino acid composition of the APHs and their different molecular weight components were determined as shown in Table 2. From the table, it is seen that in APHs, Glu is present at the highest level, followed by Asp. The amino acid compositions of APHs-1, APHs-2 and APHs-3 are similar to those of APHs, and the APHs, APHs-1, APHs-2 and APHs-3 all have high levels of hydrophobic amino acids and aromatic amino acids. Among them, APHs-1 has the highest content of hydrophobic amino acids and aromatic amino acids, which may be related to its strongest antioxidant ability, especially free radical scavenging ability.
TABLE 2 analysis of amino acid composition
(5) The APHs-1 is further separated by Sephadex G-25 as column chromatography packing, and the elution curve is shown in figure 11. Three more obvious elution peaks are detected under the wavelength of 220 nm and are named as APHs-1-a, APHs-1-b and APHs-1-c according to the peak appearance sequence. Eluting for multiple times, respectively collecting eluates of the three components, and freeze-drying in vacuum for later use.
The method comprises the following specific steps:
(a) pretreatment of Sephadex
Weighing about 50G of sephadex G-25, washing off suspended broken particles by deionized water, soaking in 500 mL of absolute ethyl alcohol for 2 h, fully washing by deionized water, heating and stirring in a boiling water bath for 6h to fully swell, cooling and packing.
(b) Column mounting
Selecting a chromatography column with the length of 100 cm and the diameter of 1.6 cm, vertically fixing the chromatography column on an iron support, pouring 1/3 column volumes of deionized water into the chromatography column, slowly pouring the swollen sephadex G-25 into the column from the top end of the column while stirring to allow the sephadex to naturally settle in the column, and simultaneously opening the bottom end of the column to allow the deionized water in the column to slowly flow out through a thin tube. The packed Sephadex column must be free of separation and air bubbles, and the gel column must be equilibrated with deionized water equivalent to 3 times the column volume at the top with 5 cm of water column.
(c) Sample application and elution
Draining or blotting the upper water column of the gel, adding 3 mL of 50 mg/mL sample solution from the top of the column, opening the bottom of the column, and adding 3 cm water column into the column when the sample solution coincides with the highest point of the gel column. The sample was then eluted with deionized water at a flow rate of 1.5 mL/min.
(d) Sample collection
Detecting the eluent under 220 nm ultraviolet wavelength, collecting the eluent with an automatic partial collector, collecting one tube every 4 min, merging the eluents corresponding to the same separation peak according to the time of the appearance of the separation peak in the absorption value curve, and measuring and comparing the in-vitro antioxidant capacity of each elution component after freeze drying.
(6) Protective effect of APHs-1-c on oxidative damage of HepG2 cells
And establishing a TBHP induced HepG2 cell oxidative damage model. The protective effect of APHs-1-c at different concentrations on HepG2 cells is shown in FIG. 12. As can be seen, the cell survival rate of the injury group added with TBHP was 51.46%, which is about half of the cell survival rate of the blank group, indicating successful modeling. In an experimental group, the protective effect of APHs-1-c on oxidative damage of a HepG2 cell induced by TBHP is dose-dependent, and the maximum cell survival rate is 89.61% when the concentration of the APHs-1-c is 800 mu m/mL. The result shows that the APHs-1-c can obviously protect HepG2 cells from oxidative damage caused by TBHP.
(7) APHs-1-c has effect of eliminating ROS in HepG2 cells
The change of intracellular active oxygen was measured by a flow cytometer using a DCFH-DA fluorescent probe, and the results are shown in FIG. 13. After oxidative damage is induced by the HepG2 cells through TBHP, the fluorescence intensity is obviously increased and is about 6 times of that of a control group, and the result shows that the oxidative stress of the HepG2 cells is induced by the TBHP to cause a large amount of accumulated active oxygen in the cells. After pretreatment of the almond peptide APHs-1-c, the fluorescence intensity in the HepG2 cell is obviously reduced along with the increase of the concentration of the almond peptide, and the almond peptide APHs-1-c has obvious dose dependence, which indicates that the almond peptide APHs-1-c can effectively eliminate the intracellular ROS generated by the oxidative stress of the HepG2 cell induced by TBHP.
(8) Effect of APHs-1-c on Keap1, Nrf2 and HO-1 expression in HepG2 cells
As shown in FIG. 14, compared with the blank group, under the action of TBHP, the Keap1 protein expression level is slightly up-regulated, and the protein expression levels of Nrf2 and HO-1 are reduced, which indicates that the HeapG2 cells are in an oxidative stress state. Compared with the injury group, the expression level of the Keap1 protein of the APHs-1-c administration group is obviously reduced, the expression levels of Nrf2 and HO-1 are obviously increased, and the dosage dependence is shown. The test result shows that the APHs-1-c can protect HepG2 cells from oxidative stress injury caused by TBHP by activating a Keap1-Nrf2/ARE pathway.
Example 4 qualitative analysis
An appropriate amount of the sample powder was redissolved with 0.15% acetic acid. Washing the polypeptide extract with 0.15% acetic acid for 2 times, desalting the filtrate with C18 StageTip, and vacuum freeze drying. After drying, the peptide fragment was reconstituted with 0.1% TFA and the peptide fragment concentration was measured at 260 nm for LC-MS/MS analysis.
(1) LC-MS/MS analysis
The result of APHs-1-c mass spectrum Basepeak is shown in figure 15 after the APHs-1-c is subjected to mass spectrum analysis and library searching qualitative analysis.
An appropriate amount of sample was taken for chromatographic separation using an EasynLC1200 chromatographic system. Buffer solution: the solution A is 0.1% formic acid, and the solution B is 0.1% formic acid and 80% acetonitrile. The column was equilibrated with 100% of liquid A. After the sample is injected into Trap Column (100 mu m multiplied by 20 mm multiplied by 5 mu m, C18), gradient separation is carried out through a chromatographic analysis Column (75 mu m multiplied by 150 mm multiplied by 3 mu m, C18), and the flow rate is 300 nl/min. The liquid phase separation gradient was as follows: 0-2 min, 2% -5% of solution B; 2-44 min, 5% -28% of solution B; 44-51 min, 28% -40% of solution B; 51-53 min, 40% -100% of liquid B; 53-60 min, 100% B liquid.
The peptide fragments were separated and analyzed by DDA (data dependent acquisition) mass spectrometry using a Q-exact HF-X mass spectrometer. The analysis time is 60 min, and the detection mode is as follows: positive ion, parent ion scan range: 350-1800 m/z, first-order mass spectrum resolution: 60,000 @ m/z 200, AGC target:3e6, first order Maximum IT: 50 ms. Peptide fragment secondary mass spectrometry was collected as follows: triggering and acquiring secondary mass spectrum maps of 20 highest-intensity parent ions after each full scan, wherein the resolution ratio of the secondary mass spectrum is as follows: 15,000 @ m/z 200, AGC target:1e5, secondary Maximum IT: 50 MS, MS2 Activation Type: HCD, Isolation window: 1.6 m/z, Normalized collisionenergy: 28.
(2) Database retrieval
The mass spectrum database retrieval software adopted by the invention is PFind; the following protein database was used: is derived from the uni Protein Database of the species almond, and has 53251 Protein sequences which are downloaded in 22/11/2021. PFind analysis parameter settings are shown in table 3.
TABLE 3 PFind analysis parameter settings
(3) The almond peptide APHs-1-c obtained by separation can improve the capacity of cells for defending oxidative stress by activating a Keap1-Nrf2/ARE signal pathway. As shown in fig. 16.
Claims (5)
1. A method for extracting antioxidant peptides APHs-1-c of pearl oil apricot is characterized by comprising the following steps:
(1) weighing defatted almond powder, placing into a beaker, adding deionized water, adjusting pH to 8, performing ultrasonic extraction, centrifuging the extract, and leaving supernatant; adjusting the pH value of the obtained supernatant to 4.5, standing, centrifuging again, collecting precipitate, washing with water, and freeze-drying the precipitate in a vacuum freeze-drying machine to obtain almond protein;
(2) weighing almond protein, dissolving in deionized water to prepare a protein solution, preheating in a water bath, then adding protease, and carrying out enzymolysis, wherein the pH value of the protein solution is kept unchanged in the process; after the enzymolysis is finished, stopping the reaction, then centrifuging, taking supernatant fluid, and carrying out vacuum freeze drying to obtain almond polypeptide powder which is marked as APHs;
(3) sequentially separating the APHs by a tangential cross flow membrane with the molecular weight cutoff of 3 kDa and 10 kDa to obtain three components, collecting the three components, freeze-drying, and respectively recording the three components as APHs-1, APHs-2 and APHs-3;
(4) separating APHs-1 by using Sephadex G-25 as column chromatography filler, and naming APHs-1-a, APHs-1-b and APHs-1-c according to the peak appearance sequence at the wavelength of 220 nm; eluting for multiple times, respectively collecting eluates of the three components, vacuum freeze drying, and collecting final target APHs-1-c.
2. The extraction method according to claim 1, wherein in the step (1), the liquid-to-material ratio of the degreased almond powder to the deionized water is 25-35: 1; the ultrasonic extraction conditions are as follows: the power is 200-; centrifuging for 20 min under the condition of 7000 r/min; the re-centrifugation is carried out for 15min under the condition of 7000 r/min.
3. The extraction method according to claim 1 or 2, wherein in the step (2), the mass fraction of the protein solution is 2%; preheating the water bath to 70 ℃; the protease is one or more of neutral protease, compound protease, alkaline protease, flavourzyme and papain; the addition amount of the enzyme is 4-6%; the temperature of the enzymolysis is 45-55 ℃, the pH value is 6.0-10.0, and the enzymolysis time is 6 h; the termination reaction is heating for 15min at 100 ℃; the centrifugation is carried out for 20 min under the condition of 7000 r/min.
4. The extraction method according to claim 1, wherein in step (3), the molecular weights of the APHs-1, APHs-2 and APHs-3 are <3 kDa, 3 kDa-10 kDa and >10 kDa, respectively.
5. The application of the pearl oil apricot antioxidant peptides APHs-1-c as claimed in claim 1 in preparing antioxidant products.
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