CN116903730A - Method for improving oxidation resistance and metal ion chelating ability of ovomucoid - Google Patents
Method for improving oxidation resistance and metal ion chelating ability of ovomucoid Download PDFInfo
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- 108010064983 Ovomucin Proteins 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 32
- 229910021645 metal ion Inorganic materials 0.000 title claims abstract description 10
- 230000003647 oxidation Effects 0.000 title abstract description 6
- 238000007254 oxidation reaction Methods 0.000 title abstract description 6
- 239000000047 product Substances 0.000 claims abstract description 70
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 26
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229910001448 ferrous ion Inorganic materials 0.000 claims abstract description 23
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 19
- 238000006206 glycosylation reaction Methods 0.000 claims abstract description 16
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229940079593 drug Drugs 0.000 claims abstract description 7
- 239000003814 drug Substances 0.000 claims abstract description 7
- 229910052742 iron Inorganic materials 0.000 claims abstract description 5
- 239000013589 supplement Substances 0.000 claims abstract description 5
- 230000002292 Radical scavenging effect Effects 0.000 claims description 48
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- 238000002376 fluorescence recovery after photobleaching Methods 0.000 claims description 20
- 108010024636 Glutathione Proteins 0.000 claims description 12
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 12
- 235000018417 cysteine Nutrition 0.000 claims description 12
- 229960003180 glutathione Drugs 0.000 claims description 12
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- 238000002360 preparation method Methods 0.000 claims description 10
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 8
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 8
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 6
- -1 ABTS free radical Chemical class 0.000 claims description 4
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 4
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- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 18
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- 238000012545 processing Methods 0.000 description 4
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 3
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- JAJWGJBVLPIOOH-IZYKLYLVSA-M sodium taurocholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 JAJWGJBVLPIOOH-IZYKLYLVSA-M 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- KMVWNDHKTPHDMT-UHFFFAOYSA-N 2,4,6-tripyridin-2-yl-1,3,5-triazine Chemical compound N1=CC=CC=C1C1=NC(C=2N=CC=CC=2)=NC(C=2N=CC=CC=2)=N1 KMVWNDHKTPHDMT-UHFFFAOYSA-N 0.000 description 1
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- HEILIGJNYTWOHU-UHFFFAOYSA-N ethanol 2-hydroxybenzoic acid Chemical compound CCO.OC(=O)C1=CC=CC=C1O HEILIGJNYTWOHU-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for improving oxidation resistance and metal ion chelating ability of ovomucoid, and belongs to the technical field of biological medicines. After the egg mucin products processed by the two unfolding auxiliary glycosylation methods are subjected to simulated gastrointestinal digestion, the digestion stability of the egg mucin is reduced, the digestion resistance problem of the egg mucin is well solved, and meanwhile, the antioxidant activity and the metal ion chelating capacity of the egg mucin products are obviously improved, and particularly, the improvement in the aspect of scavenging superoxide anion free radicals is particularly obvious; the method is simple and easy to implement, greatly improves the antioxidant activity and ferrous ion chelating activity of the high-content protein ovomucoid in the eggs, and provides a new prospect for developing antioxidant medicines and iron supplements.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for improving oxidation resistance and metal ion chelating ability of ovomucoid.
Background
Egg white has rich protein content, low price and convenient eating, and is the best source of daily high-quality protein. Egg mucin is one of the highest protein content in egg white and accounts for 11% of the total protein content in egg white. The ovomucoid has higher solubility and higher thermal stability, and the high content of the ovomucoid in eggs lays a good foundation for the development of ovomucoid-related active substances. However, current studies on ovomucoid antioxidative peptides rarely report that the analysis causes are as follows: 1. ovomucoid (pi=4.1) has an isoelectric point similar to that of ovalbumin (pi=4.3-4.5), and is difficult to purify, and many studies are inconvenient because of the lack of raw materials; (2) Egg mucin has a certain inhibition effect on trypsin and is resistant to digestion, so that less research on digestion products and less research on digestion peptide activity are required. The first difficulty has been overcome by the earlier work of the inventors, and pure ovomucoid is obtained by simple ion exchange chromatography (a separation method of high-activity ovomucoid, patent No. zl201810501744. X). Aiming at solving the problems that the egg mucin is digestion-resistant, the egg mucin peptide product is difficult to generate, the biological activity is weak and the like, the prior art still has a few reports.
Disclosure of Invention
The invention aims to provide a method for improving the antioxidant and metal ion chelating ability of ovomucoid, so as to solve the problems in the prior art, and the method is simple and easy to implement, greatly improves the antioxidant activity and ferrous ion chelating activity of the ovomucoid with high protein content in eggs, and provides a new prospect for developing antioxidant medicines and iron supplements.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a method for improving oxidation resistance and metal ion chelating ability of ovomucoid, which comprises the following steps:
adding glutathione or cysteine solution into ovomucoid solution to induce ovomucoid to be unfolded, and adding sugar to carry out glycosylation reaction.
Further, the volume ratio of the amount of the ovomucoid solution to the amount of the glutathione or cysteine solution is 1:1, a step of; wherein the concentration of the ovomucoid solution is 4mg/mL, and the concentration of the glutathione or the cysteine solution is 2mg/mL.
Further, the sugar is maltose, and the molar ratio of the maltose to the lysine residue of the ovomucoid is 10:1.
further, the glycosylation reaction condition is that the water bath reaction is carried out at 90 ℃ for 8 hours.
Further, the antioxidant capacity includes ABTS free radical, superoxide anion free radical and hydroxyl radical scavenging capacity, and FRAP antioxidant capacity; the metal ion chelating ability includes a ferrous ion chelating ability.
The invention also provides an ovomucoid product prepared by the method.
The invention also provides an application of the ovomucoid product in preparing antioxidant medicines.
The invention also provides application of the egg mucin product in preparation of an iron supplement agent.
The invention discloses the following technical effects:
after the egg mucin products processed by the two unfolding auxiliary glycosylation methods are subjected to simulated gastrointestinal digestion, the digestion stability of the egg mucin is reduced, the digestion resistance problem of the egg mucin is well solved, and meanwhile, the antioxidant activity and the metal ion chelating capacity of the egg mucin products are obviously improved, and particularly, the improvement in the aspect of scavenging superoxide anion free radicals is particularly obvious.
The method is simple and easy to implement, greatly improves the antioxidant activity and ferrous ion chelating activity of high-content protein ovomucoid in eggs, and provides a new prospect for developing antioxidant medicines and iron supplements.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a SDS-PAGE result of ovomucoid simulating gastric digestion in infants; (A) The middle pore canals 1, 2, 3 and 4 are unt-OVM, h-OVM, gly-OVM and Gsh-gly-OVM in sequence; (B) The middle pore canals 1, 2, 3 and 5 are unt-OVM, h-OVM, gly-OVM and Lcy-gly-OVM in sequence;
FIG. 2 is a SDS-PAGE result of ovomucoid mimicking gastrointestinal digestion in infants; (A) The middle pore canals 1, 2, 3 and 4 are unt-OVM, h-OVM, gly-OVM and Gsh-gly-OVM in sequence; (B) The middle pore canals 1, 2, 3 and 5 are unt-OVM, h-OVM, gly-OVM and Lcy-gly-OVM in sequence;
FIG. 3 shows SDS-PAGE results of ovomucoid mimicking human gastric digestion; (A) The middle pore canals 1, 2, 3 and 4 are unt-OVM, h-OVM, gly-OVM and Gsh-gly-OVM in sequence; (B) The middle pore canals 1, 2, 3 and 5 are unt-OVM, h-OVM, gly-OVM and Lcy-gly-OVM in sequence;
FIG. 4 shows SDS-PAGE results of ovomucoid to simulate gastrointestinal digestion in an adult; (A) The middle pore canals 1, 2, 3 and 4 are unt-OVM, h-OVM, gly-OVM and Gsh-gly-OVM in sequence; (B) The middle pore canals 1, 2, 3 and 5 are unt-OVM, h-OVM, gly-OVM and Lcy-gly-OVM in sequence;
FIG. 5 is the ABTS radical scavenging ability of an ovomucoid digestive peptide; (A) Gsh-gly-OVM and three control groups simulated ABTS radical scavenging ability of infant gastrointestinal digest; (B) Gsh-gly-OVM and three control groups mimic ABTS radical scavenging ability of adult gastrointestinal digests; (C) Lcy-gly-OVM and three control groups simulated ABTS radical scavenging ability of infant gastrointestinal digest; (D) Lcy-gly-OVM and three control groups mimic ABTS radical scavenging ability of adult gastrointestinal digests; in the figure: p <0.05;
FIG. 6 is a graph showing the superoxide anion radical scavenging capacity of an ovomucoid digestive peptide; (A) Gsh-gly-OVM and three control groups simulated superoxide anion radical scavenging capacity of infant gastrointestinal digest products; (B) Gsh-gly-OVM and three control groups mimic the superoxide anion radical scavenging capacity of adult gastrointestinal digest; (C) Lcy-gly-OVM and three control groups simulated superoxide anion radical scavenging capacity of infant gastrointestinal digest products; (D) Lcy-gly-OVM and three control groups mimic the superoxide anion radical scavenging capacity of adult gastrointestinal digest; in the figure: p <0.05;
FIG. 7 is a graph showing the hydroxyl radical scavenging capacity of an ovomucoid digestive peptide; (A) Gsh-gly-OVM and three control groups simulated the hydroxy radical scavenging ability of infant gastrointestinal digest; (B) Gsh-gly-OVM and three control groups mimic the hydroxy radical scavenging ability of adult gastrointestinal digests; (C) Lcy-gly-OVM and three control groups simulated the hydroxy radical scavenging ability of infant gastrointestinal digest; (D) Lcy-gly-OVM and three control groups mimic the hydroxy radical scavenging ability of adult gastrointestinal digests; in the figure: p <0.05;
FIG. 8 shows FRAP values for ovomucoid digestive peptides; (A) Gsh-gly-OVM and three control groups simulated FRAP values for infant gastrointestinal digests; (B) Gsh-gly-OVM and three control groups mimic FRAP values for adult gastrointestinal digests; (C) Lcy-gly-OVM and three control groups simulated FRAP values for infant gastrointestinal digests; (D) Lcy-gly-OVM and three control groups mimic FRAP values for adult gastrointestinal digests; in the figure: p <0.05;
FIG. 9 is a graph showing the ferrous ion chelating ability of ovomucoid digestive peptides; (A) Gsh-gly-OVM and three control groups mimic the ferrous ion chelating ability of infant gastrointestinal digestion products; (B) Gsh-gly-OVM and three control groups mimic the ferrous ion chelating ability of adult gastrointestinal digests; (C) Lcy-gly-OVM and three control groups mimic the ferrous ion chelating ability of infant gastrointestinal digestion products; (D) Lcy-gly-OVM and three control groups mimic the ferrous ion chelating ability of adult gastrointestinal digests; in the figure: p <0.05.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
1. Method of
1. The refolding assisted glycosylated ovomucoid product was prepared according to the following two methods, respectively
(1) Preparation of glutathione-assisted glycosylated ovomucoid product
2mg/mL of glutathione was added to a medium volume of 4mg/mL of the ovomucoid solution, maltose was added (molar ratio of sugar molecule to ovomucoid lysine residue 10:1), and the mixture was reacted in a water bath at 90℃for 8 hours to prepare glutathione-assisted glycosylated ovomucoid (Gsh-gly-OVM). The egg-like mucin was treated with the same heating conditions without glutathione, without glutathione and maltose, and the untreated egg-like mucin was used as a control (hereinafter abbreviated as gly-OVM, h-OVM and unt-OVM, respectively)
(2) Preparation of cysteine-assisted glycosylated ovomucoid product
2mg/mL of cysteine was added to a medium volume of 4mg/mL of an ovomucoid solution, maltose was added (sugar molecule to ovomucoid lysine residue ratio 10:1), and the mixture was reacted in a water bath at 90℃for 8 hours to prepare cysteine-assisted glycosylated ovomucoid (Lcy-gly-OVM). Egg mucin (h-OVM) and untreated egg mucin (unt-OVM) were treated with the same heating conditions without cysteine (gly-OVM), without cysteine and maltose as controls.
2. Analysis of digestion stability of ovomucoid product
Protocols for in vitro infant and adult gastrointestinal digestion are described in Torcello-G et al (Torcello-G vomez A, dupont D, jardin J, et al, the pattern of peptides released from dairy and egg proteins is highly dependent on the simulated digestion scenario [ J ]. Food function, 2020,11 (6): 5240-5256.) and are modified appropriately based thereon, see details 2.1 and 2.2.
2.1 in vitro simulation of gastrointestinal digestion in infants
2.1.1 in vitro simulation of infant gastric digestion
(1) Preparation of artificial gastric juice: 5 15mL centrifuge tubes were prepared and 12mL of gastric digest, 7.5. Mu.L of 0.3M CaCl, were added, respectively 2 2.4mL of pepsin solution (3125U/mL) was incubated at 37℃for 10min.
(2) 5 experimental groups of ovomucoid were prepared: unt-OVM, h-OVM, gly-OVM, gsh-gly-OVM and Lcy-gly-OVM, 15mL (protein concentration of 2 mg/mL) per tube, incubation at 37℃for 10min, pH adjustment to 3.0 with 2M HCl, and corresponding addition to the artificial gastric juice after incubation, distilled water was supplemented to a total volume of 30mL. After incubation at 37℃for 0, 15, 30, 60min, 1mL of each reaction solution was taken out, and an appropriate amount of 1M NaHCO was added 3 The reaction was stopped by adjusting the pH to 7.0 and placing in an ice bath.
2.1.2 in vitro simulation of gastrointestinal digestion in infants
(1) Preparation of artificial intestinal juice: the solution environment pH was 6.5, containing trypsin (3.5U/mg), 2mM sodium taurocholate, 0.75mM CaCl 2 2mM sodium glycodeoxycholate, incubated at 37℃for 0, 15, 30 and 60min.
(2) The products of 2.1.1 digested by stomach for 60min and neutralized pH were removed 5mL each, 4.54mL of artificial intestinal juice was added, and 35. Mu.L of 2.5mg/mL trypsin, 400. Mu.L bile salts, 25. Mu.L CaCl were added, respectively 2 After being evenly mixed, the mixture is put into a water bath kettle with the temperature of 37 ℃ for incubation, and 0.6mL of each tube is taken out at 0, 15, 30 and 60min and is put into the water bath kettle with the temperature of 100 ℃ for heating for 10min to terminate the reaction.
2.2 in vitro simulation of gastrointestinal digestion in adults
2.2.1 in vitro simulation of adult gastric digestion
(1) Preparation of artificial gastric juice: 5 15mL centrifuge tubes were prepared and 12mL of gastric digest, 7.5. Mu.L of 0.3M CaCl, were added, respectively 2 2.4mL of pepsin solution (2500U/mL) was incubated at 37℃for 10min.
(2) 5 experimental groups of ovomucoid were prepared, unt-OVM, h-OVM, gly-OVM, gsh-gly-OVM and Lcy-gly-OVM each 1After incubation at 37℃for 10min, 5mL (protein concentration 2 mg/mL) was adjusted to pH 2.0 with 2M HCl and added to the incubated artificial gastric juice, and distilled water was added to a total volume of 30mL. After incubation at 37℃for 0, 15, 30 and 60min, 1mL of sample was taken per tube, and the appropriate amount of 1M NaHCO was added 3 The pH was adjusted to 7.0 and placed in an ice bath to terminate the reaction.
2.2.2 in vitro simulation of gastrointestinal digestion in adults
(1) Preparation of artificial intestinal juice: the pH of the solution was adjusted to 6.5 containing trypsin (35U/mg), 8mM sodium taurocholate, 7.5mM CaCl 2 8mM sodium glycodeoxycholate, incubated at 37℃for 0, 15, 30 and 60min.
(2) The products of 2.2.1 digested by stomach for 60min and brought to neutral pH were each removed 5mL, 2.8mL of incubated artificial intestinal juice was added to each tube, and 350. Mu.L of trypsin (2.5 mg/mL), 1.6mL of bile salts, 250. Mu.L of CaCl were added 2 After incubation at 37℃for 0, 15, 30 and 60min, 0.6mL of sample per tube was removed and placed in a water bath at 100℃for 10min and the reaction was terminated.
Detection of digestion stability of ovomucoid samples by SDS-PAGE electrophoresis
(1) Preparation of protein sample liquid
Mixing target protein liquid with a loading buffer solution according to a ratio of 4:1 (v/v) and putting the mixture into a metal water bath kettle with the temperature of 100 ℃ for denaturation treatment for 5min.
(2) Glue compounding
The thin glass plate and the thick glass plate are put together into a holder, and a separating glue reservoir is injected therebetween. And after the liquid level reaches a proper position, adding double distilled water to perform liquid sealing and glue pressing. Standing, pouring out double distilled water after the lower glue layer is solidified, then adding concentrated glue liquid, immediately putting into a comb, and pulling out the comb for standby after the gel is solidified.
(3) Loading sample
And (3) placing the gel which is completely solidified into an electrophoresis tank, pouring the electrophoresis liquid into the electrophoresis tank to scale marks, adding 15 mu L of protein sample liquid into a gel comb hole, and adding 2 mu L of pre-dyeing marker into a first hole tank of the gel.
(4) Electrophoresis
The voltage and time were set to 80V and 30min, after which the voltage was set to 120V until the indicator stopped 1cm below the glass plate.
(5) Dyeing
Taking the gel off, putting the gel into a vessel filled with double distilled water, washing the background, adding coomassie brilliant blue R-250 staining solution, and staining on a shaker for 20min.
(6) Decoloring (decoloring)
After dyeing is finished, the gel is put into double distilled water for cleaning, decoloring liquid is added and replaced in time until decoloring is complete, the gel is replaced for 1 hour to be optimal, and then photographing is carried out in a gel imaging system.
4. Determination of antioxidant Activity of ovomucoid digestion products
4.1 determination of the ability of ABTS free radical scavenging Capacity
After 38.4mg ABTS and 6.623mg potassium persulfate are mixed, distilled water is added to a constant volume of 10mL, the light reaction is carried out at room temperature for 14-16 hours, and then distilled water is used for dilution until the absorbance value of the solution at 734nm is about 0.7+/-0.02. To 50. Mu.L of the sample solution (diluted to a concentration of 0.2 mg/mL) was added 150. Mu.L of the ABTS+ solution, and after 30min of reaction at room temperature, the absorbance was measured at 734nm in an ELISA apparatus, and the blank control used a blank solvent instead of the sample. The calculation formula is as follows:
ABTS radical scavenging ability (%) =1- (a) S /A 0 )；
Wherein:
A S : absorbance values for the sample set;
A 0 : blank absorbance values.
4.2 determination of superoxide anion radical scavenging Capacity
mu.L of the sample solution (diluted to a concentration of 0.4 mg/mL) was added to the 96-well plate, followed by adding 75 mu LTris-HCl buffer (50 mmol/L, pH 8.3), standing at 25℃for 10min, adding pyrogallic acid solution (1.5 mmol/L, solute 1.0mmol/L HCl) and reacting for 5min, and measuring absorbance at 320nm, with glutathione and cysteine as positive controls, respectively, and the blank control was a blank solvent instead of the sample. The calculation formula is as follows:
superoxide anion radical scavenging ability (%) = (a) 0 -A 1 )/(A 0 -A 2 )；
Wherein:
A 0 : absorbance values for the blank group;
A 1 : absorbance values for the sample set;
A 2 : absorbance values for positive control group.
4.3 determination of the free radical scavenging ability of hydroxyl groups
mu.L of the sample solution (diluted to a concentration of 0.8 mg/mL) was added to the 96-well ELISA plate, and 50. Mu.L of 6 mmol/LFASO was added sequentially 4 After incubation at 37℃for 10min, 50. Mu.L of 6mmol/L ethanol-salicylic acid solution was added, incubation at 37℃for 30min, and absorbance at 563nm was measured. The calculation formula is as follows:
hydroxyl radical scavenging ability (%) =1- (a) S /A 0 )；
Wherein:
A S : absorbance values for the sample set;
A 0 : blank absorbance values.
4.4FRAP Capacity determination
(1) Acetic acid buffer (0.3M, pH 3.6), 10mmol/LTPTZ solution (in 40mM HCl) and 20mmol/LFECl 3 The FRAP working solution is obtained after the solutions are mixed according to the ratio of 10:1:1.
(2) Preparing standard curve solution: feSO at 0.1-1mmol/L 4 Standard liquid replaces the sample to draw a standard curve.
(3) mu.L of the sample solution (diluted to a concentration of 1.6 mg/mL) and a standard solution were added to a 96-well plate, 250. Mu.L of FRAP working solution was added to each well, and the mixture was incubated at 37℃for 15 minutes, and the absorbance at 563nm was measured.
(4) Substituting the sample OD value into the standard curve to calculate the FRAP value.
4.5 ferrous ion chelating Capacity determination
mu.L of sample solution (diluted to a concentration of 0.2 mg/mL) and 15. Mu.L of 0.5 mmol/LFECl were taken 2 The solution was mixed, 110. Mu.L of ultrapure water was added thereto, and the mixture was left at room temperature for 2 minutes. After the reaction is finished, adding 100 mu L of Ferrozine reaction solution, fully oscillating, uniformly mixing, standing at room temperature for reaction for 10min, and then placing the reaction system into an enzyme-labeling instrument for 5minAbsorbance values were measured at 62nm and blank solvent was used instead of sample as a blank control. The calculation formula is as follows:
ferrous ion chelating ability (%) =1- (a) S /A 0 )；
Wherein:
A S : absorbance values for the sample set;
A 0 : blank absorbance values.
5. Statistical method
Experimental data were statistically analyzed using SPSS 18.0, all usingStatistical variance was determined to have statistical variance using one-way analysis of variance at p <0.05.
2. Results
1. Analysis of digestion stability of ovomucoid samples
1.1 analysis of digestion stability of ovomucoid to simulate gastric digestion in infants
To ensure good contrast effect of the electrophoresis bands of each sample, gsh-gly-OVM and Lcy-gly-OVM were combined with control unt-OVM, h-OVM and gly-OVM, respectively, at the time of loading. SDS-PAGE electrophoresis patterns simulating the infant gastric digestion of unt-OVM, h-OVM, gly-OVM, gsh-gly-OVM and Lcy-gly-OVM are shown in FIG. 1. It can be seen that within 30min of gastric digestion, the band with a molecular weight of about 28kDa did not change significantly from the pre-digestion state of each sample; at 60min of digestion, unt-OVM was indistinguishable from before digestion, whereas the bands for h-OVM, gly-OVM, gsh-gly-OVM and Lcy-gly-OVM were shallower around 28kDa than before digestion. Several processing modes are described to reduce the digestion stability of ovomucoid.
1.2 analysis of digestion stability of ovomucoid to simulate gastric digestion in infants and to simulate intestinal digestion
The electrophoretogram of the sample simulating gastric digestion of an infant for 60min, simulating intestinal digestion of an infant for various times (0, 15, 30 and 60 min) is shown in fig. 2. It was seen that the bands of h-OVM, gly-OVM, gsh-gly-OVM and Lcy-gly-OVM became progressively shallower around 28kDa with the low molecular weight enzyme products appearing below the bands, while the bands of 28kDa were not significantly different from those before digestion even after 60min of intestinal digestion of unprocessed ovomucoid. Several processing modes are described to obviously reduce the digestion stability of the ovomucoid for simulating the gastrointestinal digestion of infants.
1.3 analysis of digestion stability of ovomucoid to simulate gastric digestion in adults
SDS-PAGE electrophoresis of unt-OVM, h-OVM, gly-OVM, gsh-gly-OVM and Lcy-gly-OVM after gastric digestion of the simulated adults is shown in FIG. 3. It can be seen that within 30min of gastric digestion, the band with a molecular weight of about 28kDa was unchanged from that before digestion in each group; at 60min of digestion, unt-OVM was only slightly altered compared to before digestion, whereas the bands for h-OVM, gly-OVM, gsh-gly-OVM and Lcy-gly-OVM were significantly shallower around 28kDa than before digestion. Several processing modes are described to reduce the digestion stability of ovomucoid.
1.4 analysis of digestion stability of ovomucoid to simulate intestinal digestion after gastric digestion in adult humans
The electrophoretogram of samples simulated for 60min of adult stomach digestion simulated for various times (0, 15, 30 and 60 min) of adult intestine digestion is shown in fig. 4. It can be seen that with increasing intestinal digestion time, bands of h-OVM, gly-OVM, gsh-gly-OVM and Lcy-gly-OVM become progressively shallower around 28kDa, almost completely degraded after 30min intestinal digestion, low molecular weight enzyme digestion products appear under the bands, whereas after 60min intestinal digestion treatment of unprocessed ovomucoid, bands around 28kDa only slightly degrade, with no apparent enzyme digestion product bands appearing. Several processing modes are described to significantly reduce the digestion stability of ovomucoid to simulate gastrointestinal digestion in adults.
2. ABTS radical scavenging ability of digestive peptides
As shown in fig. 5 and tables 1 and 2, the ABTS radical scavenging ability of Gsh-gly-OVM and Lcy-gly-OVM products in the simulated infant and adult gastrointestinal digestion products was significantly increased (p < 0.05) over three control groups (unt-OVM, h-OVM, gly-OVM). Wherein the ABTS free radical scavenging ability of the Gsh-gly-OVM group simulating infant gastrointestinal digestion products is higher than unt-OVM, and the digestion products of the h-OVM group and the gly-OVM group are respectively increased by 435.73%, 132.48% and 119.99%; the Gsh-gly-OVM group mimics the ABTS radical scavenging ability of the human gastrointestinal digests by 274.36%, 94.68% and 74.15% respectively, as compared to unt-OVM, h-OVM and gly-OVM group digests. The ABTS free radical scavenging ability of the Lcy-gly-OVM group simulating infant gastrointestinal digestion products is higher than unt-OVM, and the digestion products of the h-OVM group and the gly-OVM group are respectively increased by 558.61%, 185.79% and 170.44%; the Lcy-gly-OVM group mimics the ABTS radical scavenging ability of the human gastrointestinal digests by 255.39%, 99.18% and 78.18% respectively, as compared to unt-OVM, h-OVM and gly-OVM group digests. The ABTS free radical scavenging ability of the egg mucin products obtained by the two unfolding auxiliary glycosylation methods after simulated gastrointestinal digestion is obviously higher than that of the unprocessed egg mucin and the corresponding digests of the egg mucin processed by conventional heating and glycosylation.
Table 1Gsh-gly-OVM and three control groups mimic the ABTS radical scavenging ability of infant and adult gastrointestinal digests
| Simulation of infant gastrointestinal digestion products | Simulation of gastrointestinal digestion products in adults | |
| unt-OVM | 11.43±0.71a | 24.87±0.30a |
| h-OVM | 26.33±1.40a | 44.38±0.17a |
| gly-OVM | 27.83±0.19a | 49.61±0.61a |
| Gsh-gly-OVM | 61.22±0.70 | 86.40±0.18 |
In the same column, P <0.05 compared to Gsh-gly-OVM.
Table 2Lcy-gly-OVM and three control groups mimic the ABTS radical scavenging ability of infant and adult gastrointestinal digests
| Simulation of infant gastrointestinal digestion products | Simulation of gastrointestinal digestion products in adults | |
| unt-OVM | 11.43±0.71a | 24.87±0.30a |
| h-OVM | 26.33±1.40a | 44.38±0.17a |
| gly-OVM | 27.83±0.19a | 49.61±0.61a |
| Lcy-gly-OVM | 75.26±0.13 | 88.40±0.53 |
In the same column, P <0.05 compared to Lcy-gly-OVM.
3. Superoxide anion radical scavenging ability of digestive peptides
To observe the superoxide anion radical scavenging capacity of the digested peptides, the invention adopts the pyrogallic acid method to measure, as shown in fig. 6, table 3 and table 4, the superoxide anion radical scavenging capacity of Gsh-gly-OVM and Lcy-gly-OVM products in the gastrointestinal digestion products of simulated infants and adults is significantly increased (p < 0.05) compared with three control groups (unt-OVM, h-OVM and gly-OVM). Wherein the Gsh-gly-OVM group simulates the superoxide anion radical scavenging capacity of infant gastrointestinal digestion products to be unt-OVM, and the h-OVM and gly-OVM groups digestion products respectively rise to 3220%, 253.19% and 260.87%; the Gsh-gly-OVM group simulates the superoxide anion radical scavenging capacity of human gastrointestinal digests, which is 77.43%, 214.958% and 89.25% higher than unt-OVM, and h-OVM and gly-OVM groups digests, respectively. The superoxide anion radical scavenging capacity of the infant gastrointestinal digestion products simulated by the Lcy-gly-OVM group is higher than unt-OVM, and the superoxide anion radical scavenging capacity of the infant gastrointestinal digestion products simulated by the h-OVM group and the gly-OVM group are respectively increased by 4298.95%, 368.04% and 378.24%; the Lcy-gly-OVM group simulates the superoxide anion radical scavenging capacity of human gastrointestinal digests compared with unt-OVM, and the h-OVM and gly-OVM groups digests increased 132.00%, 311.82% and 147.45%, respectively. The super-oxygen anion free radical scavenging ability of the egg mucin products obtained by the two unfolding auxiliary glycosylation methods after simulated gastrointestinal digestion is obviously higher than that of the corresponding digests of the unprocessed egg mucin by conventional heating and glycosylation treatment.
Table 3Gsh-gly-OVM and three control groups mimic the superoxide anion radical scavenging capacity of infant and adult gastrointestinal digestion products
In the same column, P <0.05 compared to Gsh-gly-OVM.
Table 4Lcy-gly-OVM and three control groups mimic the superoxide anion radical scavenging capacity of infant and adult gastrointestinal digestion products
In the same column, P <0.05 compared to Lcy-gly-OVM.
4. Hydroxyl radical scavenging ability of digestive peptides
To observe the hydroxyl radical scavenging ability of the digestive peptides, the invention uses the Fenton experiment to generate hydroxyl radical reaction method to determine, as shown in FIG. 7, table 5 and Table 6, that the hydroxyl radical scavenging ability of Gsh-gly-OVM and Lcy-gly-OVM products in the gastrointestinal digestion products of simulated infants and adults is significantly improved (p < 0.05) compared with three control groups (unt-OVM, h-OVM and gly-OVM). Wherein the Gsh-gly-OVM group simulates the hydroxy radical scavenging ability of infant gastrointestinal digestion products to be higher than unt-OVM, and the h-OVM and gly-OVM groups digestion products respectively rise by 321.58%, 244.20% and 375.96%; the Gsh-gly-OVM group mimics the hydroxyl radical scavenging capacity of the human gastrointestinal digests by 81.84%, 129.41% and 135.34% respectively, as compared to unt-OVM, h-OVM and gly-OVM group digests. The hydroxyl radical scavenging capacity of the Lcy-gly-OVM group simulated infant gastrointestinal digestion products is higher than unt-OVM, and the hydroxyl radical scavenging capacity of the h-OVM and gly-OVM group digestion products are respectively increased by 323.90%, 246.10% and 378.58%; the Lcy-gly-OVM group mimics the hydroxyl radical scavenging capacity of the human gastrointestinal digests by 162.41%, 231.05% and 239.61% respectively, as compared to unt-OVM, h-OVM and gly-OVM group digests. The hydroxy radical scavenging ability of the egg mucin products obtained by the two unfolding auxiliary glycosylation methods after simulated gastrointestinal digestion is obviously higher than that of the unprocessed egg mucin and the corresponding digests of the egg mucin processed by conventional heating and glycosylation.
Table 5Gsh-gly-OVM and three control groups mimic the hydroxy radical scavenging ability of infant and adult gastrointestinal digests
In the same column, P <0.05 compared to Gsh-gly-OVM.
Table 6Lcy-gly-OVM and three control groups mimic the hydroxy radical scavenging ability of infant and adult gastrointestinal digests
In the same column, P <0.05 compared to Lcy-gly-OVM.
5. FRAP antioxidant capacity of digestive peptides
To observe the FRAP antioxidant capacity of the digestion peptide, the invention uses a method that the antioxidant substance reduces ferrous ions in the solution and forms a blue-violet complex with TPTZ under a low pH environment. The results are shown in FIG. 8 and Table 7, table 8, and the FRAP antioxidant capacity of Gsh-gly-OVM and Lcy-gly-OVM products in the simulated infant and adult gastrointestinal digestion products is significantly increased (p < 0.05) compared with three control groups (unt-OVM, h-OVM, gly-OVM). Wherein the FRAP antioxidant capacity of the Gsh-gly-OVM group simulated infant gastrointestinal digestion products is higher than unt-OVM, and the digestion products of the h-OVM group and the gly-OVM group are respectively increased by 21.25%, 17.26% and 7.21%; the Gsh-gly-OVM group mimics the FRAP antioxidant capacity of the human gastrointestinal digests by 41.62%, 36.18% and 21.52% over unt-OVM, h-OVM and gly-OVM group digests, respectively. The FRAP antioxidant capacity of the infant gastrointestinal digestion products simulated by the Lcy-gly-OVM group is higher than that of the infant gastrointestinal digestion products simulated by the unt-OVM group, and the digestion products of the h-OVM group and the gly-OVM group are respectively increased by 48.56%, 43.66% and 31.35%; the Lcy-gly-OVM group mimics the FRAP antioxidant capacity of the human gastrointestinal digests by 66.70%, 60.31% and 43.05% over unt-OVM, h-OVM and gly-OVM group digests, respectively. The FRAP oxidation resistance of the egg mucin products obtained by the two unfolding auxiliary glycosylation methods after simulated gastrointestinal digestion is obviously higher than that of the unprocessed egg mucin and the corresponding digests of the egg mucin processed by conventional heating and glycosylation.
Table 7Gsh-gly-OVM and three control groups simulated FRAP values for infant and adult gastrointestinal digests
In the same column, P <0.05 compared to Gsh-gly-OVM.
Table 8Lcy-gly-OVM and three control groups simulated FRAP values for infant and adult gastrointestinal digests
In the same column, P <0.05 compared to Lcy-gly-OVM.
6. Ferrous ion chelating ability of digestive peptides
To observe the ferrous ion chelating ability of the digestive peptides, the invention adopts the Ferrozine method to measure, as shown in fig. 9, table 9 and table 10, the ferrous ion chelating ability of Gsh-gly-OVM and Lcy-gly-OVM products in the gastrointestinal digestion products of simulated infants and adults is significantly improved (p < 0.05) compared with three control groups (unt-OVM, h-OVM, gly-OVM). Wherein the ferrous ion chelating ability of the Gsh-gly-OVM group simulated infant gastrointestinal digestion products is higher than unt-OVM, and the h-OVM and gly-OVM group digestion products are respectively increased by 25.70%, 27.01% and 26.21%; the Gsh-gly-OVM group simulates the ferrous ion chelating ability of the human gastrointestinal digests compared with unt-OVM, and the h-OVM and gly-OVM groups digests increased by 18.43%, 30.25% and 24.06%, respectively. The ferrous ion chelating ability of the Lcy-gly-OVM group simulated infant gastrointestinal digestion products is higher than unt-OVM, and the h-OVM and gly-OVM group digestion products respectively rise by 36.37%, 37.79% and 36.92%; the Lcy-gly-OVM group mimics the ferrous ion chelating ability of the human gastrointestinal digests by 18.68%, 30.52% and 24.32% over unt-OVM, h-OVM and gly-OVM group digests, respectively. The ferrous ion chelating ability of the egg mucin products obtained by the two unfolding auxiliary glycosylation methods after simulated gastrointestinal digestion is obviously higher than that of the unprocessed egg mucin and the corresponding digests of the egg mucin processed by conventional heating and glycosylation.
Table 9Gsh-gly-OVM and three control groups mimic the ferrous ion sequestration Capacity of infant and adult gastrointestinal digestion products
In the same column, P <0.05 compared to Gsh-gly-OVM.
Table 10Lcy-gly-OVM and three control groups mimic the ferrous ion sequestration Capacity of infant and adult gastrointestinal digestion products
In the same column, P <0.05 compared to Lcy-gly-OVM.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (8)
1. A method for improving the antioxidant and metal ion sequestering capacity of ovomucoid, comprising the steps of:
adding glutathione or cysteine solution into ovomucoid solution to induce ovomucoid to be unfolded, and adding sugar to carry out glycosylation reaction.
2. The method of claim 1, wherein the volume ratio of the amount of the ovomucoid solution to the amount of the glutathione or cysteine solution is 1:1, a step of; wherein the concentration of the ovomucoid solution is 4mg/mL, and the concentration of the glutathione or the cysteine solution is 2mg/mL.
3. The method of claim 1, wherein the sugar is maltose and the molar ratio of maltose to lysine residues of the ovomucoid is 10:1.
4. the method of claim 1, wherein the glycosylation reaction is carried out in a water bath at 90 ℃ for 8 hours.
5. The method of claim 1, wherein the antioxidant capacity comprises ABTS free radical, superoxide anion free radical and hydroxyl radical scavenging capacity, and FRAP antioxidant capacity; the metal ion chelating ability includes a ferrous ion chelating ability.
6. An ovomucoid product obtained by the process of any one of claims 1-5.
7. Use of an ovomucoid product according to claim 6 for the preparation of an antioxidant drug.
8. Use of an ovomucoid product according to claim 6 for the preparation of an iron supplement.
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| JP2014171424A (en) * | 2013-03-08 | 2014-09-22 | Niigata Univ | Manufacturing method of egg white material with selectively reduced ovomucoid allergen |
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| CN103304657A (en) * | 2012-03-16 | 2013-09-18 | 东北林业大学 | Glycosylated modifying method for improving antioxidant activity of whey protein |
| JP2014171424A (en) * | 2013-03-08 | 2014-09-22 | Niigata Univ | Manufacturing method of egg white material with selectively reduced ovomucoid allergen |
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| E. D. N. S. ABEYRATHNE等: "Enzymatic hydrolysis of ovomucoid and the functional properties of its hydrolysates", POULTRY SCIENCE, vol. 94, pages 2280 - 2287 * |
| HAO JING等: "Comparison of Physicochemical and Antioxidant Properties of Egg-White Proteins and Fructose and Inulin Maillard Reaction Products", FOOD BIOPROCESS TECHNOL, vol. 4, pages 1489 - 1496, XP019961258, DOI: 10.1007/s11947-009-0279-7 * |
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