CN113264982A - Method for preparing high-oxidation-resistance collagen tripeptide-polyphenol covalent compound by using low-temperature plasma - Google Patents
Method for preparing high-oxidation-resistance collagen tripeptide-polyphenol covalent compound by using low-temperature plasma Download PDFInfo
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 19
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- 239000011347 resin Substances 0.000 claims abstract description 60
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- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims abstract description 32
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- 239000000243 solution Substances 0.000 claims description 141
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- 239000012498 ultrapure water Substances 0.000 claims description 44
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- 238000005406 washing Methods 0.000 claims description 41
- 239000000203 mixture Substances 0.000 claims description 29
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 28
- 239000007789 gas Substances 0.000 claims description 20
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 17
- 239000001301 oxygen Substances 0.000 claims description 17
- 229910052760 oxygen Inorganic materials 0.000 claims description 17
- 238000004108 freeze drying Methods 0.000 claims description 16
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- 239000012535 impurity Substances 0.000 claims description 14
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 claims description 12
- 238000012545 processing Methods 0.000 claims description 12
- 238000009832 plasma treatment Methods 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 8
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 claims description 7
- 235000005487 catechin Nutrition 0.000 claims description 7
- 238000011068 loading method Methods 0.000 claims description 7
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- 108090000765 processed proteins & peptides Proteins 0.000 claims description 7
- 238000005070 sampling Methods 0.000 claims description 7
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- 229940074391 gallic acid Drugs 0.000 claims description 6
- 235000004515 gallic acid Nutrition 0.000 claims description 6
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 claims description 5
- WMBWREPUVVBILR-WIYYLYMNSA-N (-)-Epigallocatechin-3-o-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=C(O)C=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-WIYYLYMNSA-N 0.000 claims description 5
- WMBWREPUVVBILR-UHFFFAOYSA-N GCG Natural products C=1C(O)=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-UHFFFAOYSA-N 0.000 claims description 5
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- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 8
- 230000003078 antioxidant effect Effects 0.000 description 5
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- 150000001413 amino acids Chemical class 0.000 description 3
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- 230000031700 light absorption Effects 0.000 description 2
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 2
- -1 polyphenol compounds Chemical class 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
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- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention discloses a method for preparing a collagen tripeptide-polyphenol covalent compound with high oxidation resistance by using low-temperature plasma, which comprises the following steps: 1. dynamic high-pressure micro-jet premixing; 2. electrolytic water treatment; 3. preparing a collagen tripeptide-polyphenol covalent compound by using low-temperature plasma; 4. primarily separating AB-8 macroporous resin; 5. two-step separation of Sephadex LH-20; 6. concentrating the covalent compound under reduced pressure; 7. covalent Compound Dry powder preparation. The collagen tripeptide and the polyphenol are treated by dynamic high-pressure microjet, so that the mixing uniformity of the collagen tripeptide and the polyphenol is improved; preparing an electrolyzed water treatment solution to provide conditions for covalent reaction of the electrolyzed water treatment solution and the electrolyzed water treatment solution; the collagen tripeptide-polyphenol covalent compound is prepared by adopting a low-temperature plasma technology, so that the reaction time is shortened, and the binding rate of the collagen tripeptide and the polyphenol is greatly improved; the collagen tripeptide-polyphenol covalent compound is separated and purified by two steps of AB-8 macroporous resin and Sephadex LH-20, the purity of the compound is obviously improved, and the biological activity of the compound is kept.
Description
Technical Field
The invention relates to a processing technology of collagen tripeptide, in particular to a method for preparing a collagen tripeptide-polyphenol covalent compound with high oxidation resistance by using low-temperature plasma.
Background
The collagen tripeptide is an active substance which is formed by hydrolyzing collagen to a certain degree and consists of three amino acids and has a special sequence (Gly-A-B). In the amino acid sequence Gly-a-B of the collagen tripeptide, a is typically proline or hydroxyproline, while B may be any amino acid. The collagen tripeptide has the effects of resisting oxidation, maintaining beauty, keeping young, preventing aging and the like. The small molecule collagen tripeptide has small molecular weight, and can be directly absorbed by the small intestine without being digested after being ingested by a human body, so that the biological activity of the small molecule collagen tripeptide is retained. The polyphenol compounds refer to plant extracts containing a plurality of phenolic hydroxyl groups in the structure, and can be divided into apple polyphenol, mulberry polyphenol, blueberry polyphenol, tea polyphenol and the like according to the sources. Polyphenols in foods are a major source of antioxidants for humans.
The low-temperature plasma technology is a non-thermal food processing technology which utilizes high pressure, rays or electromagnetic field to process gas and leads the gas to generate collision ionization to form plasma. The plasma includes many excited electrons, ions, atoms, radicals, etc. that provide activation energy for chemical reactions. At present, in the field of food, low-temperature plasma is mainly used for sterilization treatment of food, and there is no report on promoting covalent reaction of collagen tripeptide and polyphenol by using low-temperature plasma.
Disclosure of Invention
The invention aims to provide a method for preparing a high-oxidation-resistance collagen tripeptide-polyphenol covalent compound by using low-temperature plasma, which takes collagen tripeptide as a raw material, adds polyphenol and then adopts dynamic high-pressure microjet to fully and uniformly mix the solution; treating the mixed solution with an electrolyzed water preparation apparatus to generate hydroxyl ions; treating a mixed solution of collagen tripeptide and polyphenol by taking oxygen as experimental gas of low-temperature plasma to prepare a collagen tripeptide-polyphenol covalent compound; separating and purifying the covalent compound by two steps by using AB-8 macroporous resin and a Sephadex LH-20 chromatographic column; finally, preparing the collagen tripeptide-polyphenol covalent compound after decompression concentration and freeze drying. Thereby greatly improving the antioxidant activity of the collagen tripeptide.
In order to achieve the purpose, the invention provides a method for preparing a collagen tripeptide-polyphenol covalent compound with high oxidation resistance by using low-temperature plasma, which comprises the following step 1. dynamic high-pressure microjet premixing
Taking collagen tripeptide as a raw material, adding polyphenol according to a proportion, primarily mixing, and preparing a uniform mixture of the collagen tripeptide and the polyphenol under the dynamic high-pressure microjet treatment;
2. electrolytic water treatment
Preparing the mixed solution of the step 1 by using electrolyzed water;
3. preparation of collagen tripeptide-polyphenol covalent compound by low-temperature plasma
Collecting the mixture of collagen tripeptide and polyphenol obtained in the step 2, and treating the mixture by low-temperature plasma to prepare a collagen tripeptide-polyphenol covalent compound;
primary separation with AB-8 macroporous resin
Pre-treating AB-8 macroporous resin, loading a column and balancing, then sampling the solution obtained in the step (3), eluting collagen peptides which are not subjected to covalent bonding by using distilled water, eluting polyphenol which is bonded with collagen tripeptide and is not bonded by using an ethanol acid solution, collecting the eluent for the second time, and concentrating the eluent obtained for the second time by using a rotary evaporator under reduced pressure;
sephadex LH-20 two-step separation
Pre-treating Sephadex LH-20, packing column, balancing, and separating the concentrated eluate in step 4, eluting unbound polyphenol with high-concentration ethanol solution, and eluting collagen tripeptide bound with polyphenol with medium-concentration ethanol solution;
6. concentration of covalent Compounds under reduced pressure
Concentrating the collagen tripeptide-polyphenol covalent compound solution obtained in the step 5 under reduced pressure by using a rotary evaporator;
7. dry powder preparation of covalent Compounds
And (4) freeze-drying the concentrated solution obtained in the step (6) by using a freeze dryer to prepare collagen tripeptide-polyphenol covalent compound dry powder.
In the step 1, the addition amount of polyphenol is 5-15 parts relative to 90 parts of collagen tripeptide.
In the step 1, the polyphenol is catechin, gallic acid and epigallocatechin gallate, and the use ratio of the three polyphenols is 1-5: 1-5: 1 to 5.
In the step 1, the dynamic high-pressure micro-jet treatment pressure is 50-100 MPa, and the treatment time is 1-3 min.
The treatment time of the electrolyzed water preparation instrument in the step 2 is 3-8 min, and the treatment voltage is 3-10V.
The experimental gas of the low-temperature plasma in the step 3 is oxygen; the oxygen supply amount is 50-100%; the low-temperature plasma processing power is 100-200W; the low-temperature plasma treatment time is 3-5 min.
The pretreatment conditions of the AB-8 macroporous resin in the step 4 are as follows: and (5) soaking and washing with ultrapure water for 24-48 h to remove impurities and fully swelling.
Filling the AB-8 macroporous resin in the step 4 into a chromatographic column, and washing with 2-3 times of 95% ethanol solution of the macroporous resin; washing with ultrapure water until the column is free of ethanol; washing with 1-2 times of 3-5% NaOH solution of macroporous resin; washing the column with ultrapure water without NaOH; washing with 1-2 times of 3-5% HCl solution of macroporous resin; the column was washed with ultrapure water and then freed from HCl.
The AB-8 macroporous resin column in the step 4 is balanced by distilled water; in the step 4, the acid solution of the ethanol is a 95% ethanol solution which contains 0.3-05% hydrochloric acid; and 4, concentrating under reduced pressure until the volume of the concentrated solution is 2-5 mL.
In the step 5, the Sephadex LH-20 pretreatment condition is that the mixture is soaked and washed by ultrapure water for 24-48 h to remove impurities and is fully swelled; the Sephadex LH-20 equilibrium conditions are: balancing to a stable baseline by using ultrapure water; in the step 5, the ethanol solution with medium concentration is 40-50% ethanol solution, and the ethanol solution with high concentration is 80-95% ethanol solution.
In the step 6, the solution is decompressed and concentrated to a volume of 10-20% of the original volume; and (7) freeze-drying the concentrated solution in the step (7) for 48 hours to obtain collagen tripeptide-polyphenol covalent compound dry powder.
Drawings
FIG. 1 is a diagram showing the mechanism of covalent bonding between tripeptide and polyphenol of collagen according to the present invention; hydroxyl radicals attack amino groups or sulfhydryl groups of the collagen tripeptide to form an intermediate product, and the formed intermediate product is covalently bonded with polyphenol to generate a collagen tripeptide-polyphenol covalent compound.
Detailed Description
The technical solution of the present invention is further described in detail by the following examples.
The invention discloses a method for preparing a collagen tripeptide-polyphenol covalent compound with high oxidation resistance by using low-temperature plasma, which comprises the following steps:
1. dynamic high pressure microjet premixing
Taking collagen tripeptide as a raw material, adding polyphenol according to a proportion, primarily mixing, and preparing a uniform mixture of the collagen tripeptide and the polyphenol under the dynamic high-pressure microjet treatment;
2. electrolytic water treatment
Treating the mixed solution in the step 1 by using an electrolytic water preparation instrument;
3. preparation of collagen tripeptide-polyphenol covalent compound by low-temperature plasma
Collecting the mixture of collagen tripeptide and polyphenol obtained in the step 2, and treating the mixture by low-temperature plasma to prepare a collagen tripeptide-polyphenol covalent compound;
primary separation with AB-8 macroporous resin
Pre-treating AB-8 macroporous resin, loading a column and balancing, then sampling the solution obtained in the step (3), eluting collagen peptides which are not subjected to covalent bonding by using distilled water, eluting polyphenol which is bonded with collagen tripeptide and is not bonded by using an ethanol acid solution, collecting the eluent for the second time, and concentrating the eluent obtained for the second time by using a rotary evaporator under reduced pressure;
sephadex LH-20 two-step separation
And (4) pretreating Sephadex LH-20, filling a column, and carrying out secondary separation on the eluate concentrated in the step 4 after balancing. Firstly, the collagen tripeptide combined with polyphenol is eluted by ethanol solution with medium concentration, then the unbound polyphenol is eluted by ethanol solution with high concentration,
6. concentration of covalent Compounds under reduced pressure
Concentrating the collagen tripeptide-polyphenol covalent compound solution obtained in the step 5 under reduced pressure by using a rotary evaporator;
7. dry powder preparation of covalent Compounds
And (4) freeze-drying the concentrated solution obtained in the step (6) by using a freeze dryer to prepare collagen tripeptide-polyphenol covalent compound dry powder.
In the step 1, the addition amount of polyphenol is 5-15 parts relative to 90 parts of collagen tripeptide.
In the step 1, the polyphenol comprises catechin, gallic acid and epigallocatechin gallate, and the use ratio of the three components is 1-5: 1-5: 1 to 5.
In the step 1, the dynamic high-pressure micro-jet treatment pressure is 60MPa, and the treatment time is 2 min.
The treatment time of the electrolyzed water preparation apparatus in the step 2 is 5min, and the treatment voltage is 5V.
The experimental gas of the low-temperature plasma in the step 3 is oxygen.
And 3, the supply amount of the experimental gas oxygen of the low-temperature plasma in the step 3 is 50-100%.
And 3, the low-temperature plasma processing power is 100-200W.
And 3, the low-temperature plasma treatment time in the step 3 is 3-5 min.
The pretreatment conditions of the AB-8 macroporous resin in the step 4 are as follows: and soaking and washing with ultrapure water for 24h to remove impurities and fully swell.
Step 4, filling the AB-8 macroporous resin into a chromatographic column, and washing with 2 times of ethanol (95%) solution of the macroporous resin; washing with ultrapure water until the column is free of ethanol; washing with 1 time of 4% NaOH solution of macroporous resin; washing the column with ultrapure water without NaOH; washing with 1 time of 4% HCl solution of macroporous resin; the column was washed with ultrapure water and then freed from HCl.
And (4) balancing the AB-8 macroporous resin column by using distilled water in the step 4.
The acid solution of ethanol in step 4 is a 95% ethanol solution containing 0.3% hydrochloric acid.
And 4, concentrating under reduced pressure until the volume of the concentrated solution is 3 mL.
In the step 5, the pretreatment condition of Sephadex LH-20 is that the mixture is soaked and washed by ultrapure water for 24 hours to remove impurities and fully swell.
The Sephadex LH-20 equilibrium conditions in step 5 are as follows: equilibrate to baseline with ultrapure water.
The ethanol solution with the medium concentration in the step 5 is a 50% ethanol solution.
The high-concentration ethanol solution in the step 5 is an 80% ethanol solution.
And 6, concentrating the solution under reduced pressure until the volume of the solution is 10 percent of the original volume.
And (7) freeze-drying the concentrated solution in the step (7) for 48 hours to obtain collagen tripeptide-polyphenol covalent compound dry powder.
Examples one to nine
Experiments show that the low-temperature plasma treatment power, the treatment time and the supply of experimental gas oxygen in the step 2 have great influence on the antioxidant activity of the collagen tripeptide-polyphenol covalent compound. Therefore, the following orthogonal experiment is designed by selecting the three conditions, and reaction conditions are optimized by changing the parameter values of low-temperature plasma processing power, processing time and processing gas. Nine sets of tests are designed, orthogonal experiment optimization is carried out on three factors of low-temperature plasma treatment power, treatment time and treatment gas, and other processes of the nine sets of tests, such as dynamic high-pressure microjet premixing, electrolytic water treatment, AB-8 macroporous resin primary separation, Sephadex LH-20 two-step separation, freeze drying and the like, are the same.
And (3) antioxidant determination: the antioxidant capacity of collagen tripeptide-polyphenol covalent compounds was measured as dpph.clearance. 0.2mmol/L DPPH solution was prepared from methanol solution, and 500. mu.L DPPH solution was added with the same volume of collagen tripeptide-polyphenol covalent compound solution. Reacting at room temperature for 45min (in dark place), and measuring light absorption value (A) at 517nms). The control group (A) was prepared by using 500. mu.L of methanol to replace collagen tripeptide-polyphenol covalent compoundc) A reaction system in which 500. mu.L of ultrapure water was used in place of DPPH solution was a sample blank (A)b) A blank set (A) was prepared by replacing the collagen tripeptide-polyphenol covalent compound and DPPH solution with 500. mu.L of methanol and ultrapure water, respectively0). The experiment was repeated three times. The DPPH-clearance calculation formula for collagen tripeptide-polyphenol covalent compounds is as follows:
table 1 experimental design and results
From the results of the orthogonal experiments, it was found that the main factors affecting the antioxidant activity of the collagen tripeptide-polyphenol covalent compound were low temperature plasma treatment power and treatment gas, and then reaction time, and the optimal conditions for preparing the collagen tripeptide-polyphenol covalent compound were treatment power of 200W, reaction time of 4min, and treatment gas of 100% oxygen.
Example ten, comprising the steps of:
1. dynamic high pressure microjet premixing
Taking collagen tripeptide as a raw material, adding polyphenol according to a proportion, primarily mixing, and preparing a uniform mixture of the collagen tripeptide and the polyphenol under the dynamic high-pressure microjet treatment;
2. electrolytic water treatment
Treating the mixed solution in the step 1 by using an electrolytic water preparation instrument;
3. preparation of collagen tripeptide-polyphenol covalent compound by low-temperature plasma
Collecting the mixture of collagen tripeptide and polyphenol obtained in the step 2, and treating the mixture by low-temperature plasma to prepare a collagen tripeptide-polyphenol covalent compound;
primary separation with AB-8 macroporous resin
Pre-treating AB-8 macroporous resin, loading a column and balancing, then sampling the solution obtained in the step (3), eluting collagen peptides which are not subjected to covalent bonding by using distilled water, eluting polyphenol which is bonded with collagen tripeptide and is not bonded by using an ethanol acid solution, collecting the eluent for the second time, and concentrating the eluent obtained for the second time by using a rotary evaporator under reduced pressure;
sephadex LH-20 two-step separation
And (4) pretreating Sephadex LH-20, filling a column, and carrying out secondary separation on the eluate concentrated in the step 4 after balancing. Firstly, the collagen tripeptide combined with polyphenol is eluted by ethanol solution with medium concentration, then the unbound polyphenol is eluted by ethanol solution with high concentration,
6. concentration of covalent Compounds under reduced pressure
Concentrating the collagen tripeptide-polyphenol covalent compound solution obtained in the step 5 under reduced pressure by using a rotary evaporator;
7. dry powder preparation of covalent Compounds
And (4) freeze-drying the concentrated solution obtained in the step (6) by using a freeze dryer to prepare collagen tripeptide-polyphenol covalent compound dry powder.
In step 1, the polyphenol is added in an amount of 15 parts relative to 90 parts of the collagen tripeptide.
In the step 1, the polyphenol comprises catechin, gallic acid and epigallocatechin gallate, and the use ratio of the three components is 3: 5: 7.
in the step 1, the dynamic high-pressure micro-jet treatment pressure is 60MPa, and the treatment time is 2 min.
The treatment time of the electrolyzed water preparation apparatus in the step 2 is 5min, and the treatment voltage is 5V.
The experimental gas of the low-temperature plasma in the step 3 is oxygen.
The oxygen supply of the experimental gas of the low-temperature plasma in the step 3 is 100%.
And 3, the low-temperature plasma processing power is 200W.
The low-temperature plasma treatment time in step 3 is 4 min.
The pretreatment conditions of the AB-8 macroporous resin in the step 4 are as follows: and soaking and washing with ultrapure water for 24h to remove impurities and fully swell.
Step 4, filling the AB-8 macroporous resin into a chromatographic column, and washing with 2 times of ethanol (95%) solution of the macroporous resin; washing with ultrapure water until the column is free of ethanol; washing with 1 time of 4% NaOH solution of macroporous resin; washing the column with ultrapure water without NaOH; washing with 1 time of 4% HCl solution of macroporous resin; the column was washed with ultrapure water and then freed from HCl.
And (4) balancing the AB-8 macroporous resin column by using distilled water in the step 4.
The acid solution of ethanol in step 4 is a 95% ethanol solution containing 0.3% hydrochloric acid.
And 4, concentrating under reduced pressure until the volume of the concentrated solution is 3 mL.
In the step 5, the pretreatment condition of Sephadex LH-20 is that the mixture is soaked and washed by ultrapure water for 24 hours to remove impurities and fully swell.
The Sephadex LH-20 equilibrium conditions in step 5 are as follows: equilibrate to baseline with ultrapure water.
The ethanol solution with the medium concentration in the step 5 is a 50% ethanol solution.
The high-concentration ethanol solution in the step 5 is an 80% ethanol solution.
And 6, concentrating the solution under reduced pressure until the volume of the solution is 10 percent of the original volume.
And (7) freeze-drying the concentrated solution in the step (7) for 48 hours to obtain collagen tripeptide-polyphenol covalent compound dry powder.
And (3) determining the binding rate: measuring the content of free amino in the sample by using (o-phthalaldehyde) OPA solution, and calculating the binding rate of the covalent compound, wherein the calculation formula of the binding rate is as follows:
wherein, ODmaxIs the absorbance, OD, of the collagen tripeptide solutionsIs the corresponding light absorption value of the collagen tripeptide-polyphenol covalent compound.
EXAMPLE eleven
1. Dynamic high pressure microjet premixing
Taking collagen tripeptide as a raw material, adding polyphenol according to a proportion, primarily mixing, and preparing a uniform mixture of the collagen tripeptide and the polyphenol under the dynamic high-pressure microjet treatment;
2. electrolytic water treatment
Treating the mixed solution in the step 1 by using an electrolytic water preparation instrument;
3. preparation of collagen tripeptide-polyphenol covalent compound by low-temperature plasma
Collecting the mixture of collagen tripeptide and polyphenol obtained in the step 2, and treating the mixture by low-temperature plasma to prepare a collagen tripeptide-polyphenol covalent compound;
primary separation with AB-8 macroporous resin
Pre-treating AB-8 macroporous resin, loading a column and balancing, then sampling the solution obtained in the step (3), eluting collagen peptides which are not subjected to covalent bonding by using distilled water, eluting polyphenol which is bonded with collagen tripeptide and is not bonded by using an ethanol acid solution, collecting the eluent for the second time, and concentrating the eluent obtained for the second time by using a rotary evaporator under reduced pressure;
sephadex LH-20 two-step separation
And (4) pretreating Sephadex LH-20, filling a column, and carrying out secondary separation on the eluate concentrated in the step 4 after balancing. Firstly, the collagen tripeptide combined with polyphenol is eluted by ethanol solution with medium concentration, then the unbound polyphenol is eluted by ethanol solution with high concentration,
6. concentration of covalent Compounds under reduced pressure
Concentrating the collagen tripeptide-polyphenol covalent compound solution obtained in the step 5 under reduced pressure by using a rotary evaporator;
7. dry powder preparation of covalent Compounds
And (4) freeze-drying the concentrated solution obtained in the step (6) by using a freeze dryer to prepare collagen tripeptide-polyphenol covalent compound dry powder.
In step 1, the polyphenol is added in an amount of 15 parts relative to 90 parts of the collagen tripeptide.
In the step 1, the polyphenol comprises catechin, gallic acid and epigallocatechin gallate, and the use ratio of the three components is 3: 5: 7.
in the step 1, the dynamic high-pressure micro-jet treatment pressure is 60MPa, and the treatment time is 2 min.
The treatment time of the electrolyzed water preparation apparatus in the step 2 is 5min, and the treatment voltage is 5V.
The experimental gas of the low-temperature plasma in the step 3 is oxygen.
The oxygen supply of the experimental gas of the low-temperature plasma in the step 3 is 60%.
And 3, the low-temperature plasma processing power is 100W.
The low-temperature plasma treatment time in step 3 is 4 min.
The pretreatment conditions of the AB-8 macroporous resin in the step 4 are as follows: and soaking and washing with ultrapure water for 24h to remove impurities and fully swell.
Step 4, filling the AB-8 macroporous resin into a chromatographic column, and washing with 2 times of ethanol (95%) solution of the macroporous resin; washing with ultrapure water until the column is free of ethanol; washing with 1 time of 4% NaOH solution of macroporous resin; washing the column with ultrapure water without NaOH; washing with 1 time of 4% HCl solution of macroporous resin; the column was washed with ultrapure water and then freed from HCl.
And (4) balancing the AB-8 macroporous resin column by using distilled water in the step 4.
The acid solution of ethanol in step 4 is a 95% ethanol solution containing 0.3% hydrochloric acid.
And 4, concentrating under reduced pressure until the volume of the concentrated solution is 3 mL.
In the step 5, the pretreatment condition of Sephadex LH-20 is that the mixture is soaked and washed by ultrapure water for 24 hours to remove impurities and fully swell.
The Sephadex LH-20 equilibrium conditions in step 5 are as follows: equilibrate to baseline with ultrapure water.
The ethanol solution with the medium concentration in the step 5 is a 50% ethanol solution.
The high-concentration ethanol solution in the step 5 is an 80% ethanol solution.
And 6, concentrating the solution under reduced pressure until the volume of the solution is 10 percent of the original volume.
And (7) freeze-drying the concentrated solution in the step (7) for 48 hours to obtain collagen tripeptide-polyphenol covalent compound dry powder.
Example twelve
1. Dynamic high pressure microjet premixing
Taking collagen tripeptide as a raw material, adding polyphenol according to a proportion, primarily mixing, and preparing a uniform mixture of the collagen tripeptide and the polyphenol under the dynamic high-pressure microjet treatment;
2. electrolytic water treatment
Treating the mixed solution in the step 1 by using an electrolytic water preparation instrument;
3. preparation of collagen tripeptide-polyphenol covalent compound by low-temperature plasma
Collecting the mixture of collagen tripeptide and polyphenol obtained in the step 2, and treating the mixture by low-temperature plasma to prepare a collagen tripeptide-polyphenol covalent compound;
primary separation with AB-8 macroporous resin
Pre-treating AB-8 macroporous resin, loading a column and balancing, then sampling the solution obtained in the step (3), eluting collagen peptides which are not subjected to covalent bonding by using distilled water, eluting polyphenol which is bonded with collagen tripeptide and is not bonded by using an ethanol acid solution, collecting the eluent for the second time, and concentrating the eluent obtained for the second time by using a rotary evaporator under reduced pressure;
sephadex LH-20 two-step separation
And (4) pretreating Sephadex LH-20, filling a column, and carrying out secondary separation on the eluate concentrated in the step 4 after balancing. Firstly, the collagen tripeptide combined with polyphenol is eluted by ethanol solution with medium concentration, then the unbound polyphenol is eluted by ethanol solution with high concentration,
6. concentration of covalent Compounds under reduced pressure
Concentrating the collagen tripeptide-polyphenol covalent compound solution obtained in the step 5 under reduced pressure by using a rotary evaporator;
7. dry powder preparation of covalent Compounds
And (4) freeze-drying the concentrated solution obtained in the step (6) by using a freeze dryer to prepare collagen tripeptide-polyphenol covalent compound dry powder.
In step 1, the polyphenol is added in an amount of 15 parts relative to 90 parts of the collagen tripeptide.
The polyphenols in step 1 comprise only catechins.
In the step 1, the dynamic high-pressure micro-jet treatment pressure is 60MPa, and the treatment time is 2 min.
The treatment time of the electrolyzed water preparation apparatus in the step 2 is 5min, and the treatment voltage is 5V.
The experimental gas of the low-temperature plasma in the step 3 is oxygen.
The oxygen supply of the experimental gas of the low-temperature plasma in the step 3 is 60%.
And 3, the low-temperature plasma processing power is 100W.
The low-temperature plasma treatment time in step 3 is 4 min.
The pretreatment conditions of the AB-8 macroporous resin in the step 4 are as follows: and soaking and washing with ultrapure water for 24h to remove impurities and fully swell.
Step 4, filling the AB-8 macroporous resin into a chromatographic column, and washing with 2 times of ethanol (95%) solution of the macroporous resin; washing with ultrapure water until the column is free of ethanol; washing with 1 time of 4% NaOH solution of macroporous resin; washing the column with ultrapure water without NaOH; washing with 1 time of 4% HCl solution of macroporous resin; the column was washed with ultrapure water and then freed from HCl.
And (4) balancing the AB-8 macroporous resin column by using distilled water in the step 4.
The acid solution of ethanol in step 4 is a 95% ethanol solution containing 0.3% hydrochloric acid.
And 4, concentrating under reduced pressure until the volume of the concentrated solution is 3 mL.
In the step 5, the pretreatment condition of Sephadex LH-20 is that the mixture is soaked and washed by ultrapure water for 24 hours to remove impurities and fully swell.
The Sephadex LH-20 equilibrium conditions in step 5 are as follows: equilibrate to baseline with ultrapure water.
The ethanol solution with the medium concentration in the step 5 is a 50% ethanol solution.
The high-concentration ethanol solution in the step 5 is an 80% ethanol solution.
And 6, concentrating the solution under reduced pressure until the volume of the solution is 10 percent of the original volume.
And (7) freeze-drying the concentrated solution in the step (7) for 48 hours to obtain collagen tripeptide-polyphenol covalent compound dry powder.
EXAMPLE thirteen
1. Dynamic high pressure microjet premixing
Taking collagen tripeptide as a raw material, adding polyphenol according to a proportion, primarily mixing, and preparing a uniform mixture of the collagen tripeptide and the polyphenol under the dynamic high-pressure microjet treatment;
2. electrolytic water treatment
Treating the mixed solution in the step 1 by using an electrolytic water preparation instrument;
3. preparation of collagen tripeptide-polyphenol covalent compound by low-temperature plasma
Collecting the mixture of collagen tripeptide and polyphenol obtained in the step 2, and treating the mixture by low-temperature plasma to prepare a collagen tripeptide-polyphenol covalent compound;
primary separation with AB-8 macroporous resin
Pre-treating AB-8 macroporous resin, loading a column and balancing, then sampling the solution obtained in the step (3), eluting collagen peptides which are not subjected to covalent bonding by using distilled water, eluting polyphenol which is bonded with collagen tripeptide and is not bonded by using an ethanol acid solution, collecting the eluent for the second time, and concentrating the eluent obtained for the second time by using a rotary evaporator under reduced pressure;
sephadex LH-20 two-step separation
And (4) pretreating Sephadex LH-20, filling a column, and carrying out secondary separation on the eluate concentrated in the step 4 after balancing. Firstly, the collagen tripeptide combined with polyphenol is eluted by ethanol solution with medium concentration, then the unbound polyphenol is eluted by ethanol solution with high concentration,
6. concentration of covalent Compounds under reduced pressure
Concentrating the collagen tripeptide-polyphenol covalent compound solution obtained in the step 5 under reduced pressure by using a rotary evaporator;
7. dry powder preparation of covalent Compounds
And (4) freeze-drying the concentrated solution obtained in the step (6) by using a freeze dryer to prepare collagen tripeptide-polyphenol covalent compound dry powder.
In step 1, the polyphenol is added in an amount of 15 parts relative to 90 parts of the collagen tripeptide.
The polyphenols in step 1 include catechins and gallic acid. The ratio of use was such that 7: 8.
in the step 1, the dynamic high-pressure micro-jet treatment pressure is 60MPa, and the treatment time is 2 min.
The treatment time of the electrolyzed water preparation apparatus in the step 2 is 5min, and the treatment voltage is 5V.
The experimental gas of the low-temperature plasma in the step 3 is oxygen.
The oxygen supply of the experimental gas of the low-temperature plasma in the step 3 is 80%.
And 3, the low-temperature plasma processing power is 200W.
The low-temperature plasma treatment time in step 3 is 4 min.
The pretreatment conditions of the AB-8 macroporous resin in the step 4 are as follows: and soaking and washing with ultrapure water for 24h to remove impurities and fully swell.
Step 4, filling the AB-8 macroporous resin into a chromatographic column, and washing with 2 times of ethanol (95%) solution of the macroporous resin; washing with ultrapure water until the column is free of ethanol; washing with 1 time of 4% NaOH solution of macroporous resin; washing the column with ultrapure water without NaOH; washing with 1 time of 4% HCl solution of macroporous resin; the column was washed with ultrapure water and then freed from HCl.
And (4) balancing the AB-8 macroporous resin column by using distilled water in the step 4.
The acid solution of ethanol in step 4 is a 95% ethanol solution containing 0.3% hydrochloric acid.
And 4, concentrating under reduced pressure until the volume of the concentrated solution is 3 mL.
In the step 5, the pretreatment condition of Sephadex LH-20 is that the mixture is soaked and washed by ultrapure water for 24 hours to remove impurities and fully swell.
The Sephadex LH-20 equilibrium conditions in step 5 are as follows: equilibrate to baseline with ultrapure water.
The ethanol solution with the medium concentration in the step 5 is a 50% ethanol solution.
The high-concentration ethanol solution in the step 5 is an 80% ethanol solution.
And 6, concentrating the solution under reduced pressure until the volume of the solution is 10 percent of the original volume.
And (7) freeze-drying the concentrated solution in the step (7) for 48 hours to obtain collagen tripeptide-polyphenol covalent compound dry powder.
The DPPH clearance results for the collagen tripeptide-polyphenol covalent compounds prepared in examples ten to thirteen are shown in table two.
TABLE DPPH clearance and Polyphenol binding
| Experimental group | DPPH·(％) | Binding Rate (%) |
| Example ten | 84.53 | 34.33 |
| EXAMPLE eleven | 79.58 | 26.54 |
| Example twelve | 80.19 | 27.75 |
| EXAMPLE thirteen | 83.47 | 30.92 |
The foregoing merely represents preferred embodiments of the invention, which are described in some detail and detail, and therefore should not be construed as limiting the scope of the invention. It should be noted that, for those skilled in the art, various changes, modifications and substitutions can be made without departing from the spirit of the present invention, and these are all within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for preparing a collagen tripeptide-polyphenol covalent compound with high oxidation resistance by using low-temperature plasma is characterized by comprising the following steps:
1. dynamic high pressure microjet premixing
Taking collagen tripeptide as a raw material, adding polyphenol according to a proportion, primarily mixing, and preparing a uniform mixture of the collagen tripeptide and the polyphenol under the dynamic high-pressure microjet treatment;
2. electrolytic water treatment
Preparing the mixed solution of the step 1 by using electrolyzed water;
3. preparation of collagen tripeptide-polyphenol covalent compound by low-temperature plasma
Collecting the mixture of collagen tripeptide and polyphenol obtained in the step 2, and treating the mixture by low-temperature plasma to prepare a collagen tripeptide-polyphenol covalent compound;
primary separation with AB-8 macroporous resin
Pre-treating AB-8 macroporous resin, loading a column and balancing, then sampling the solution obtained in the step (3), eluting collagen peptides which are not subjected to covalent bonding by using distilled water, eluting polyphenol which is bonded with collagen tripeptide and is not bonded by using an ethanol acid solution, collecting the eluent for the second time, and concentrating the eluent obtained for the second time by using a rotary evaporator under reduced pressure;
sephadex LH-20 two-step separation
Pre-treating Sephadex LH-20, packing column, balancing, and separating the concentrated eluate in step 4, eluting unbound polyphenol with high-concentration ethanol solution, and eluting collagen tripeptide bound with polyphenol with medium-concentration ethanol solution;
6. concentration of covalent Compounds under reduced pressure
Concentrating the collagen tripeptide-polyphenol covalent compound solution obtained in the step 5 under reduced pressure by using a rotary evaporator;
7. dry powder preparation of covalent Compounds
And (4) freeze-drying the concentrated solution obtained in the step (6) by using a freeze dryer to prepare collagen tripeptide-polyphenol covalent compound dry powder.
2. The method according to claim 1, wherein the polyphenol is added in an amount of 5 to 15 parts per 90 parts of the collagen tripeptide in step 1, wherein the polyphenol is catechin, gallic acid and epigallocatechin gallate in step 1, and the ratio of the three polyphenols used is 1 to 5: 1-5: 1 to 5.
3. The method according to claim 1, wherein the dynamic high-pressure micro-jet treatment pressure in step 1 is 50-100 MPa, and the treatment time is 1-3 min.
4. The method according to claim 1, wherein the treatment time of the electrolyzed water forming apparatus in the step 2 is 3 to 8min, and the treatment voltage is 3 to 10V.
5. The method of claim 1, wherein the experimental gas of the low temperature plasma in step 3 is oxygen; the oxygen supply amount is 50-100%; the low-temperature plasma processing power is 100-200W; the low-temperature plasma treatment time is 3-5 min.
6. The method of claim 1, wherein the AB-8 macroporous resin pretreatment conditions in step 4 are: and (5) soaking and washing with ultrapure water for 24-48 h to remove impurities and fully swelling.
7. The method according to claim 1, wherein in the step 4, the AB-8 macroporous resin is filled into a chromatographic column and then is washed by 2-3 times of 95% ethanol solution of the macroporous resin; washing with ultrapure water until the column is free of ethanol; washing with 1-2 times of 3-5% NaOH solution of macroporous resin; washing the column with ultrapure water without NaOH; washing with 1-2 times of 3-5% HCl solution of macroporous resin; the column was washed with ultrapure water and then freed from HCl.
8. The method of claim 1, wherein the AB-8 macroporous resin column in step 4 is equilibrated with distilled water; in the step 4, the acid solution of the ethanol is a 95% ethanol solution which contains 0.3-05% hydrochloric acid; and 4, concentrating under reduced pressure until the volume of the concentrated solution is 2-5 mL.
9. The method as claimed in claim 1, wherein the Sephadex LH-20 pretreatment condition in the step 5 is that the mixture is washed with ultrapure water for 24-48 h to remove impurities and fully swelled; the Sephadex LH-20 equilibrium conditions are: balancing to a stable baseline by using ultrapure water; in the step 5, the ethanol solution with medium concentration is 40-50% ethanol solution, and the ethanol solution with high concentration is 80-95% ethanol solution.
10. The method according to claim 1, wherein in the step 6, the solution is concentrated under reduced pressure to 10-20% of the original volume; and (7) freeze-drying the concentrated solution in the step (7) for 48 hours to obtain collagen tripeptide-polyphenol covalent compound dry powder.
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