CN116837062A - Chinese torreya seed peptide and preparation method and application thereof - Google Patents
Chinese torreya seed peptide and preparation method and application thereof Download PDFInfo
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- CN116837062A CN116837062A CN202310893935.6A CN202310893935A CN116837062A CN 116837062 A CN116837062 A CN 116837062A CN 202310893935 A CN202310893935 A CN 202310893935A CN 116837062 A CN116837062 A CN 116837062A
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- torreya
- enzymolysis
- seed peptide
- torreya grandis
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- 240000000147 Torreya grandis Species 0.000 title claims abstract description 96
- 235000016410 Torreya grandis Nutrition 0.000 title claims abstract description 96
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 69
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000006228 supernatant Substances 0.000 claims abstract description 22
- 239000000047 product Substances 0.000 claims abstract description 18
- 108090000145 Bacillolysin Proteins 0.000 claims abstract description 11
- 102000035092 Neutral proteases Human genes 0.000 claims abstract description 11
- 108091005507 Neutral proteases Proteins 0.000 claims abstract description 11
- 108091005658 Basic proteases Proteins 0.000 claims abstract description 10
- 108090000526 Papain Proteins 0.000 claims abstract description 10
- 239000004365 Protease Substances 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 10
- 235000019834 papain Nutrition 0.000 claims abstract description 10
- 229940055729 papain Drugs 0.000 claims abstract description 10
- 230000001105 regulatory effect Effects 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 9
- 238000001914 filtration Methods 0.000 claims abstract description 9
- 241000488908 Torreya Species 0.000 claims abstract description 8
- 239000012535 impurity Substances 0.000 claims abstract description 8
- 235000013305 food Nutrition 0.000 claims abstract description 5
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 230000007935 neutral effect Effects 0.000 claims abstract description 5
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 4
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 4
- 239000002537 cosmetic Substances 0.000 claims abstract description 4
- 238000004108 freeze drying Methods 0.000 claims abstract description 4
- 239000011259 mixed solution Substances 0.000 claims abstract description 4
- 230000002087 whitening effect Effects 0.000 claims abstract description 4
- 230000002378 acidificating effect Effects 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 25
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 229940088598 enzyme Drugs 0.000 claims description 14
- 239000000843 powder Substances 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 108090000790 Enzymes Proteins 0.000 claims description 13
- 239000003208 petroleum Substances 0.000 claims description 12
- 230000000415 inactivating effect Effects 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 9
- 238000000967 suction filtration Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 5
- 238000009835 boiling Methods 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 238000002835 absorbance Methods 0.000 description 15
- -1 hydroxyl radicals Chemical class 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 7
- 102000004196 processed proteins & peptides Human genes 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 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 6
- 230000000694 effects Effects 0.000 description 6
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 5
- 230000002292 Radical scavenging effect Effects 0.000 description 4
- 102000003425 Tyrosinase Human genes 0.000 description 4
- 108060008724 Tyrosinase Proteins 0.000 description 4
- 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 4
- 238000002474 experimental method Methods 0.000 description 4
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 description 3
- 230000007760 free radical scavenging Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000000643 oven drying Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 230000002000 scavenging effect Effects 0.000 description 2
- 244000147058 Derris elliptica Species 0.000 description 1
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 1
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 1
- 241001116495 Taxaceae Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000002792 antioxidant assay Methods 0.000 description 1
- 239000010692 aromatic oil Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/14—Drugs for dermatological disorders for baldness or alopecia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- 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 relates to a torreya grandis seed peptide and a preparation method and application thereof, and the method comprises the following steps: mixing the Chinese torreya seeds after impurity removal with water to obtain a mixed solution, adjusting the pH to be alkaline, adding alkaline protease for enzymolysis, centrifuging, and collecting a first supernatant; adjusting the pH of the first supernatant to be neutral, adding neutral protease for enzymolysis, centrifuging, and collecting a second supernatant; regulating the pH value of the second supernatant to be acidic, adding papain for enzymolysis, centrifuging, collecting the third supernatant, and freeze-drying to obtain a torreya seed peptide enzymolysis product; filtering the enzymolysis product of Chinese torreya seed peptide, collecting components with molecular weight less than 3-5kDa, and drying to obtain Chinese torreya seed peptide. Compared with the prior art, the torreya seed peptide prepared by the invention has stronger antioxidant and whitening activities, and promotes the application of torreya seeds in the industries of foods, medicines and cosmetics.
Description
Technical Field
The invention relates to the technical field of polypeptide preparation, in particular to a torreya grandis seed peptide and a preparation method and application thereof.
Background
Chinese Torreya (Torreya grandis 'Merrilli'), a perennial evergreen arbor of Torreya of Taxaceae, is a special woody oil tree species in our country, and has long cultivation and eating history. The Chinese torreya seed is rich in unsaturated fatty acid, protein and carbohydrate, has more than 20 aromatic components, and is a natural high-quality raw material of high-grade aromatic oil and extract.
Researches on Chinese torreya seeds mainly focus on the influence on the quality of oil and aroma components of the Chinese torreya seeds, and the application scene of the Chinese torreya seeds mainly comprises two modes of processing into nut foods and squeezing into Chinese torreya seed oil. At present, little research is done on bioactive components such as proteins, carbohydrates and the like with low content in Chinese torreya seeds. Therefore, the types of the processed products are single, the finish and deep processed products are lacked, the industrialization level is low, and the utilization rate and the added value of the Chinese torreya seeds are low. The nutritional ingredients in the torreya grandis seeds are lack of full utilization, and a certain resource waste is caused.
Proteins are the material basis of life, are formed by connecting amino acids in a dehydration condensation mode, are also essential nutrients for human bodies, and have various biological activities. However, the molecular weight of proteins is tens of thousands, even hundreds of thousands and millions, and the proteins are difficult to be directly absorbed and utilized by human bodies. The polypeptide is used as a functional active fragment forming protein, has a molecular structure between amino acid and protein, is easy to be absorbed and utilized by human bodies, has various biological activities such as antioxidation, antibiosis, blood pressure reduction, anti-tumor, immunoregulation and the like, gradually becomes a research hot spot at home and abroad, and provides a new thought for the fine and deep processing of Chinese torreya seeds.
Disclosure of Invention
The invention aims to provide a torreya grandis seed peptide for improving the performance of the torreya grandis seed peptide, and a preparation method and application thereof.
The aim of the invention can be achieved by the following technical scheme:
one of the technical schemes of the invention is to provide a preparation method of torreya grandis seed peptide, which comprises the following steps:
s1, mixing Chinese torreya seed powder with petroleum ether for reaction, adding ethanol for reaction after primary suction filtration and drying, and performing secondary suction filtration and drying to obtain Chinese torreya seeds after impurity removal;
s2, mixing the torreya grandis seeds subjected to impurity removal in the step S1 with water to obtain a mixed solution, regulating pH to be alkaline, adding alkaline protease for enzymolysis, centrifuging, and collecting a first supernatant;
s3, regulating the pH value of the first supernatant obtained in the step S2 to be neutral, adding neutral protease for enzymolysis, and collecting a second supernatant after centrifugation;
s4, adjusting the pH value of the second supernatant obtained in the step S3 to be acidic, adding papain for enzymolysis, centrifuging, collecting a third supernatant, and freeze-drying the third supernatant to obtain a torreya grandis seed peptide enzymolysis product;
s5, filtering the torreya grandis seed peptide enzymolysis product obtained in the step S4, collecting components with molecular weight smaller than 3-5kDa, and drying to obtain torreya grandis seed peptide.
Further, in some embodiments, in step S1, the mass ratio of the torreya grandis seed powder to the petroleum ether is 1: (5-10), petroleum ether with the boiling point of 30-60 ℃ is selected as petroleum ether, and the reaction conditions of the torreya seed powder and the petroleum ether are as follows: the reaction is carried out at 25 ℃ for 6 to 10 hours.
Further, in some embodiments, in step S1, the mass ratio of torreya grandis seed powder to ethanol is 1: (5-10), ethanol with the mass fraction of 95% is selected as ethanol, and the conditions for the reaction of the product obtained by once suction filtration and drying and the ethanol are as follows: reacting for 3-6h at 60-80 ℃.
Further, in some embodiments, in step S2, the mass ratio of torreya grandis seed to water after the impurity removal in step S1 is 1: (8-20), regulating the pH to be 7.5-8.5, wherein the mass percentage of alkaline protease to the substrate is 2-5%, and the enzymolysis reaction conditions are as follows: stirring in water bath at 45-55deg.C for enzymolysis for 2-4h, and inactivating enzyme in water bath at 95-100deg.C for 15-25min.
Further, in some embodiments, in step S3, the pH is adjusted to be neutral to 6.5-7.5, the mass percent of neutral protease to substrate is 2-5%, and the conditions for the enzymatic hydrolysis reaction are: stirring in water bath at 45-55deg.C for enzymolysis for 2-4h, and inactivating enzyme in water bath at 95-100deg.C for 15-25min.
Further, in some embodiments, in step S4, the pH is adjusted to an acid of 5.5-6.5, the mass percent of papain to substrate is 2-5%, and the conditions for the enzymatic hydrolysis reaction are: stirring in water bath at 45-55deg.C for enzymolysis for 2-4h, and inactivating enzyme in water bath at 95-100deg.C for 15-25min.
Further, in some embodiments, in step S5, the filtration membrane used for filtration is an ultrafiltration membrane having a molecular weight cut-off of 3-5 kDa.
The second technical scheme of the invention is to provide a torreya grandis seed peptide, which is based on the preparation method of the torreya grandis seed peptide in one of the technical schemes.
The third technical scheme of the invention is to provide the application of the torreya grandis seed peptide in the second technical scheme, and the application of the torreya grandis seed peptide in preparing antioxidant or whitening products.
Further, in some embodiments, the products include food, pharmaceutical, and cosmetic products.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the torreya grandis seed peptide is prepared by grading through a three-enzyme compounding method for the first time, three different enzymes are selected for enzymolysis reaction under respective specific enzymolysis conditions (pH, enzymolysis temperature and enzymolysis time), so that the torreya grandis seed protein is more fully degraded, the obtained torreya grandis seed peptide has stronger antioxidant and whitening activities, and the application of torreya grandis seeds in the industries of foods, medicines and cosmetics is promoted.
Drawings
FIG. 1 is a graph showing the effect of Torreya grandis seed peptide on DPPH radical scavenging in example 1.
FIG. 2 is a graph showing the effect of Torreya grandis seed peptide on scavenging ABTS free radicals in example 1.
FIG. 3 is a graph showing the effect of Torreya grandis seed peptide on scavenging hydroxyl radicals in example 1.
FIG. 4 is a graph showing the inhibitory effect of Torreya grandis seed peptide on tyrosinase activity in example 1.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples. The present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following examples.
In the following examples and comparative examples, unless otherwise specified, the starting materials or processing techniques are all those which are conventional commercially available in the art.
The alkaline protease used in the examples below was 200U/mg in specification, the neutral protease was 200U/mg in specification, and the papain was 800U/mg in specification.
Example 1:
a preparation method of torreya grandis seed peptide comprises the following steps:
(1) Pretreatment of raw materials: breaking torreya grandis seeds into powder, and sieving with 80 mesh sieve for standby.
(2) Removing impurities: extracting the Chinese torreya seed powder with petroleum ether with boiling point of 30-60 deg.C (mass ratio of Chinese torreya seed powder to petroleum ether is 1:9) at 25deg.C for 8 hr, vacuum filtering, oven drying, adding 95% ethanol (mass ratio of Chinese torreya seed powder to 95% ethanol) at 70deg.C, extracting for 5 hr, vacuum filtering, and oven drying to obtain Chinese torreya seed powder with liposoluble and alcohol soluble components removed.
(3) Alkaline protease enzymolysis: adding deionized water into the Chinese torreya seed powder obtained in the step (1) according to the mass ratio of 1:10, regulating the pH of the mixed solution to 8.0, then adding alkaline protease (E/S=4% (w/w), E is enzyme and S is substrate), stirring in a water bath at 50 ℃ for enzymolysis for 3h, inactivating enzyme in a water bath at 95 ℃ for 20min, centrifuging at 8000r/min for 20min, and collecting supernatant.
(4) Neutral protease enzymolysis: and (3) regulating the pH of the supernatant obtained in the step (3) to 7.0, adding neutral protease (E/S=3% (w/w), E is enzyme, S is substrate), stirring in a water bath at 50 ℃ for enzymolysis for 2.5h, inactivating enzyme in a water bath at 95 ℃ for 20min, centrifuging at 8000r/min for 20min, and collecting the supernatant.
(5) Papain enzymolysis: regulating the pH of the supernatant obtained in the step (4) to 6.2, adding papain (E/S=4% (w/w), E is enzyme, S is substrate), stirring in a water bath at 50 ℃ for enzymolysis for 3h, inactivating enzyme in a water bath at 95 ℃ for 20min, centrifuging at 8000r/min for 20min, collecting the supernatant, and freeze-drying to obtain the torreya seed enzymolysis product.
(6) Ultrafiltering the enzymolysis product of Chinese torreya seed, passing through ultrafiltration membrane with molecular weight cutoff of 3kDa, collecting component with molecular weight less than 3kDa, and vacuum freeze drying to obtain Chinese torreya seed peptide.
The prepared torreya grandis seed peptide is subjected to the following activity measurement:
(1) Antioxidant Activity assay:
(1-1) DPPH radical scavenging experiment
Preparation of 500. Mu.L of Torreya grandis seed peptide aqueous solution (0.625 mg/mL, 1.25mg/mL, 2.5mg/mL, 5mg/mL, 10 mg/mL) with different concentrations, adding an equal amount of 500. Mu.L of 0.2mM DPPH solution, shaking uniformly, standing for 25min in dark place, and measuring absorbance at wavelength 517 nm. The clearance rate calculation formula is:
DPPH radical clearance (%) = (1- (a) 1 -A 2 )/A 0 )×100；
Wherein A is 0 Absorbance value of the blank, A 1 Is the absorbance value of torreya grandis seed peptides with different concentrations, A 2 The background absorbance value of the torreya grandis seed peptide is measured by using ultrapure water instead of DPPH solution.
As shown in figure 1, the Chinese torreya seed peptide has better DPPH free radical scavenging capability, and the DPPH free radical scavenging rate of the Chinese torreya seed peptide reaches 94.4% at the concentration of 10 mg/mL.
(1-2) ABTS radical scavenging experiments
100. Mu.L of Torreya grandis seed peptide aqueous solutions (0.625 mg/mL, 1.25mg/mL, 2.5mg/mL, 5mg/mL, 10 mg/mL) of different concentrations were prepared, 2.9mL of ABTS solution was added, and the absorbance at a wavelength of 734nm was measured. The clearance rate calculation formula is:
ABTS radical clearance (%) = (1- (a) 1 -A 2 )/A 0 )×100；
Wherein A is 0 Absorbance value of the blank, A 1 Is the absorbance value of torreya grandis seed peptides with different concentrations, A 2 The background absorbance value of the torreya grandis seed peptide is measured by using ultrapure water instead of ABTS solution.
As shown in figure 2, the torreya grandis seed peptide has better ability of removing ABTS free radical, and the removal rate of the torreya grandis seed peptide to the ABTS free radical reaches 88.5% at the concentration of 10 mg/mL.
(1-3) hydroxyl radical scavenging experiments
Preparing 500 mu L of Torreya grandis seed peptide water solution (0.625 mg/mL, 1.25mg/mL, 2.5mg/mL, 5mg/mL, 10 mg/mL) with different concentrations, adding 200 mu L of FeSO 4 (6 mM), salicylic acid (6 mM) and H 2 O 2 (6 mM), shaking well, heating at 37℃for 30min, and measuring absorbance at 510 nm. The clearance rate calculation formula is:
hydroxyl radical removal (%) = (1- (a) 1 -A 2 )/A 0 )×100；
Wherein A is 0 Absorbance value of the blank, A 1 Is the absorbance value of torreya grandis seed peptides with different concentrations, A 2 The background absorbance value of the torreya grandis seed peptide is measured by using ultrapure water instead of ABTS solution.
As shown in figure 3, the Chinese torreya seed peptide has better capability of removing hydroxyl radicals, and the removal rate of the Chinese torreya seed peptide on the hydroxyl radicals reaches 95.6% at the concentration of 10 mg/mL.
(2) Inhibition tyrosinase activity assay
mu.L of 0.5 mmol.L -1 L-DOPA) was placed in test tubes and 200. Mu.L of each of the different concentrations was addedTorreya grandis seed peptide water solution (2 mg/mL, 4mg/mL, 6mg/mL, 8mg/mL, 10 mg/mL), water bath at 37deg.C for 10min, adding 200 μL 40 μg m L -1 Is put into a water bath at 37 ℃ for 10min. The absorbance value is measured at the wavelength 734nm, and the clearance rate calculation formula is:
tyrosinase inhibition rate (%) = (1-a) 1 /A 0 )×100；
Wherein A is 0 Absorbance value of the blank group, A 1 Is the absorbance value of the torreya grandis seed peptide groups with different concentrations.
As shown in figure 4, the torreya grandis seed peptide has certain tyrosinase inhibition capability and is concentration-dependent, and the torreya grandis seed peptide is continuously enhanced along with the increase of concentration within a certain range.
Comparative example 1:
the vast majority of the differences compared to example 1 are that the neutral protease enzymatic hydrolysis process of step (4) is omitted.
Comparative example 2:
the vast majority of the differences compared to example 1 are that the alkaline protease enzymatic hydrolysis of step (3) and the papain enzymatic hydrolysis of step (5) are omitted.
The ABTS free radical scavenging experiments were performed on the Torreya grandis seed peptides (10 mg/mL) prepared in example 1, comparative example 1 and comparative example 2, and the results are shown in Table 1.
TABTS radical clearance for different Torreya grandis seed peptides
As can be seen from the results in Table 1, the ABTS free radical clearance of the torreya grandis seed peptide prepared in example 1 reaches 88.5%, which is obviously higher than that of the torreya grandis seed peptide prepared in comparative example 1, and therefore, the ABTS free radical clearance of the torreya grandis seed peptide prepared in example 1 can be obviously improved in the enzymolysis process of neutral protease, and is also higher than that of the torreya grandis seed peptide prepared in comparative example 2, and the ABTS free radical clearance of the torreya grandis seed peptide prepared in example 1 can be obviously improved in the combined enzymolysis process of alkaline protease, neutral protease and papain.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. The preparation method of the torreya grandis seed peptide is characterized by comprising the following steps of:
s1, mixing Chinese torreya seed powder with petroleum ether for reaction, adding ethanol for reaction after primary suction filtration and drying, and performing secondary suction filtration and drying to obtain Chinese torreya seeds after impurity removal;
s2, mixing the torreya grandis seeds subjected to impurity removal in the step S1 with water to obtain a mixed solution, regulating pH to be alkaline, adding alkaline protease for enzymolysis, centrifuging, and collecting a first supernatant;
s3, regulating the pH value of the first supernatant obtained in the step S2 to be neutral, adding neutral protease for enzymolysis, and collecting a second supernatant after centrifugation;
s4, adjusting the pH value of the second supernatant obtained in the step S3 to be acidic, adding papain for enzymolysis, centrifuging, collecting a third supernatant, and freeze-drying the third supernatant to obtain a torreya grandis seed peptide enzymolysis product;
s5, filtering the torreya grandis seed peptide enzymolysis product obtained in the step S4, collecting components with molecular weight smaller than 3-5kDa, and drying to obtain torreya grandis seed peptide.
2. The preparation method of the torreya grandis seed peptide according to claim 1, wherein in the step S1, the mass ratio of torreya grandis seed powder to petroleum ether is 1: (5-10), petroleum ether with the boiling point of 30-60 ℃ is selected as petroleum ether, and the reaction conditions of the torreya seed powder and the petroleum ether are as follows: the reaction is carried out at 25 ℃ for 6 to 10 hours.
3. The preparation method of the torreya grandis seed peptide according to claim 1, wherein in the step S1, the mass ratio of torreya grandis seed powder to ethanol is 1: (5-10), ethanol with the mass fraction of 95% is selected as ethanol, and the conditions for the reaction of the product obtained by once suction filtration and drying and the ethanol are as follows: reacting for 3-6h at 60-80 ℃.
4. The preparation method of the torreya grandis seed peptide according to claim 1, wherein in the step S2, the mass ratio of torreya grandis seeds to water after impurity removal in the step S1 is 1: (8-20), regulating the pH to be 7.5-8.5, wherein the mass percentage of alkaline protease to the substrate is 2-5%, and the enzymolysis reaction conditions are as follows: stirring in water bath at 45-55deg.C for enzymolysis for 2-4h, and inactivating enzyme in water bath at 95-100deg.C for 15-25min.
5. The preparation method of torreya grandis seed peptide according to claim 1, wherein in step S3, the pH is adjusted to be neutral to 6.5-7.5, the mass percentage of neutral protease to substrate is 2-5%, and the conditions of enzymolysis reaction are: stirring in water bath at 45-55deg.C for enzymolysis for 2-4h, and inactivating enzyme in water bath at 95-100deg.C for 15-25min.
6. The method for preparing Chinese torreya seed peptide according to claim 1, wherein in step S4, the pH is adjusted to be 5.5-6.5, the mass percentage of papain to the substrate is 2-5%, and the conditions for the enzymolysis reaction are: stirring in water bath at 45-55deg.C for enzymolysis for 2-4h, and inactivating enzyme in water bath at 95-100deg.C for 15-25min.
7. The method for preparing torreya grandis seed peptide according to claim 1, wherein in step S5, the filtration membrane used for filtration is an ultrafiltration membrane with a molecular weight cut-off of 3-5 kDa.
8. A torreya grandis seed peptide, characterized in that it is based on a process for the preparation of a torreya grandis seed peptide according to any one of claims 1-7.
9. The use of the torreya grandis seed peptide according to claim 8 for the preparation of antioxidant or whitening products.
10. The use of a torreya seed peptide according to claim 9, wherein the product comprises food, pharmaceutical and cosmetic products.
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