LU500630B1 - Preparation method of fish scale gelatin high antioxidant peptide - Google Patents
Preparation method of fish scale gelatin high antioxidant peptide Download PDFInfo
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- LU500630B1 LU500630B1 LU500630A LU500630A LU500630B1 LU 500630 B1 LU500630 B1 LU 500630B1 LU 500630 A LU500630 A LU 500630A LU 500630 A LU500630 A LU 500630A LU 500630 B1 LU500630 B1 LU 500630B1
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- 241000251468 Actinopterygii Species 0.000 title claims abstract description 94
- 229920000159 gelatin Polymers 0.000 title claims abstract description 57
- 235000019322 gelatine Nutrition 0.000 title claims abstract description 57
- 108010010803 Gelatin Proteins 0.000 title claims abstract description 55
- 239000008273 gelatin Substances 0.000 title claims abstract description 55
- 235000011852 gelatine desserts Nutrition 0.000 title claims abstract description 55
- 101800000068 Antioxidant peptide Proteins 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 239000012588 trypsin Substances 0.000 claims abstract description 20
- 102000004142 Trypsin Human genes 0.000 claims abstract description 16
- 108090000631 Trypsin Proteins 0.000 claims abstract description 16
- 108091005658 Basic proteases Proteins 0.000 claims abstract description 12
- 241000252230 Ctenopharyngodon idella Species 0.000 claims abstract description 10
- 239000002994 raw material Substances 0.000 claims abstract description 10
- 239000002253 acid Substances 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 19
- 239000000047 product Substances 0.000 claims description 15
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 10
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 9
- 229910052698 phosphorus Inorganic materials 0.000 claims description 9
- 239000011574 phosphorus Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 244000025254 Cannabis sativa Species 0.000 claims description 4
- 230000003078 antioxidant effect Effects 0.000 claims description 4
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 4
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 3
- 239000003963 antioxidant agent Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000002203 pretreatment Methods 0.000 claims 1
- 235000010354 butylated hydroxytoluene Nutrition 0.000 abstract description 8
- 238000000034 method Methods 0.000 abstract description 7
- 238000012545 processing Methods 0.000 abstract description 6
- 102000008186 Collagen Human genes 0.000 abstract description 3
- 108010035532 Collagen Proteins 0.000 abstract description 3
- 229920001436 collagen Polymers 0.000 abstract description 3
- 238000007254 oxidation reaction Methods 0.000 abstract description 3
- 230000002000 scavenging effect Effects 0.000 abstract description 3
- SPSPIUSUWPLVKD-UHFFFAOYSA-N 2,3-dibutyl-6-methylphenol Chemical compound CCCCC1=CC=C(C)C(O)=C1CCCC SPSPIUSUWPLVKD-UHFFFAOYSA-N 0.000 abstract description 2
- 239000002537 cosmetic Substances 0.000 abstract description 2
- 238000004090 dissolution Methods 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 235000013402 health food Nutrition 0.000 abstract description 2
- 231100000252 nontoxic Toxicity 0.000 abstract description 2
- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- 230000003647 oxidation Effects 0.000 abstract description 2
- 238000004108 freeze drying Methods 0.000 description 6
- 239000006227 byproduct Substances 0.000 description 5
- 239000008399 tap water Substances 0.000 description 5
- 235000020679 tap water Nutrition 0.000 description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108010009736 Protein Hydrolysates Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 239000013505 freshwater Substances 0.000 description 3
- 239000000413 hydrolysate Substances 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 238000000967 suction filtration Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000001828 Gelatine Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000013401 experimental design Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000010993 response surface methodology Methods 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 208000031295 Animal disease Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000009189 diving Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002366 mineral element Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 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
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
Classifications
-
- 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
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/04—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from fish or other sea animals
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/10—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from hair, feathers, horn, skins, leather, bones, or the like
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
- A23J3/06—Gelatine
-
- 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
-
- 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
- A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
- A23L29/281—Proteins, e.g. gelatin or collagen
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Zoology (AREA)
- Nutrition Science (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Marine Sciences & Fisheries (AREA)
- Dispersion Chemistry (AREA)
- Microbiology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses preparation method of fish scale gelatin high antioxidant peptide, which decalcifies the fish scales, and then adopts alkaline protease and trypsin to carry out two-step enzymolysis on the fish scales to obtain the fish scale gelatin high antioxidant peptide. According to the invention, fish scales from grass carp processing industry are used as raw materials, the invention adopts the method of acid decalcification that accelerates the dissolution of collagen and improves the purity of antioxidant peptides, and it uses alkaline protease for one-step enzymolysis, and then uses trypsin for two-step enzymolysis to increase the content of antioxidant peptides. The invention is simple to realize, and non-toxic, the prepared antioxidant peptide has higher OH scavenging capacity than dibutylhydroxytoluene (BHT) with good oxidation resistance, and high applicable value in the fields of health food, medicine, cosmetics and the like.
Description
Description LU500630 Preparation method of fish scale gelatin high antioxidant peptide The invention belongs to the technical field of fish gelatin matenals, and particularly relates to preparation method of fish scale gelatin high antioxidant peptide.
China has a vast water area and shundant freshwater fish resources. In recent years, with the continuous increase of freshwater fish production, the freshwater fish processing industry has also increased. However, with the rapid development of the processing of minced fish and other aquatio products, a large amount of by-products will also be generated. Studies have shown that these by-products contain a lof of nutritional active substances such as protein, fat and mineral elements, but the by-products have been simply processed into feed or treated as waste, which not only causes waste of resources, economic losses, but also pollutes the environment Fish scale is one of the main by- products in the processing of freshwaler fish, which contains a lot of nufrients such as collagen. Because the surface of fish scale is attached by hydroxyapatite and other minerais, fish scale has not been effectively utilized.
in the process of food processing, in order to improve the quality of products or prolong the shelf He of goods, # is common to add certain food additives {such as dioutyihydroxyioiuens). Although they can effectively remove free radicals, slow down oxidation reaction and prevent food from rancidity, their safely factor is low, and there is also a risk of carcinogenic to human health, Antimadant peptide, which is obtained by enzymatic hydrolysis of protein, has the ability of scavenging oxygen fres radicals in the body and can play the role of anti-oxidation and anti-aging, and has become a research focus. Nowadays, due to animal diseases such as mad cow disease, swine fever, religious beliefs and the distary needs of vegstanans, the sources of mammalian polypeptides are limited, and people gradually turn their attention to aquatic animals.
This invention provides a preparation method of fish scale gelatin high antioxidant peptide that uses fish scales as raw material, and decaloifies the fish scales, then adopts alkaline protease and trypsin to make two-step enzymatic hydrolysis on fish scales, which obtains the fish scale gelatin peptide with high antioxidant activity.
Further, the preparation method comprises the slaps as below:
{1} Pre-trestment of raw materials: Remove impurities on the surface of fish scales, rinse and dry, then decalcify the fish scales with acid solution, rinse and dry the decalcified fish scales.
{2} Preparation of grass carp scale gelatine Add the dried fish scales into water, heat it for extraction, filter the mixture to obtain exiract, and fresze-dry the exiract to obtain freeze-dried fish phosphorus gelatin (3) Gne-step enzymolysis: Dissolve the freeze-dried fish phosphorus gelatin in distilled water to oblain a fish scale gelatin solution, carry out one-step enzymolysis treatment on the fish scale gelatin solution by adopting alkaline protease, inactivats the enzyme after enzymolysis, centrifugaliy separate the enzymolysis solution, and fresze- dry supernatant to obtain freeze-dried enzymuolysis products, (4y Two-step enzymolysis: Prepare the freeze-dried enzymoiysis product into an enzymolysis product solution, carry out two-step enzymaolysis with trypsin, inactivate the enzyme after enzymolysis, and centrifuge Io take supernatant to obtain the fish scale gelatin high antioxidant peptide.
Further, the acid solution is © 3-0 7moÿL hydrochloric acid, the ratio of fish scales to hydrochloric acid is 1.10~30 aim, and the decalcification time is 1 — 3h.
Further, the ratio of material to liquid is 1:15~20g:mi, the pH is 5.0~6.0, the exiraction is carried out at 80°C in water bath for more than 2 hours, and the pH is adjusted by dripping dilule hydrochloric acid into water.
Further, the mass fraction of fish phosphorus gelatin in the fish scale gelatin solution is 10%, the addition amount of alkaline protease is 1% of the mass of fish scale gelatin in the fish scale gelatin solution, the one-step enzymolysis conditions are pH 8.0, enzymolysis temperature is 50°C, enzymolysia time is 4h, and pH is adjusted to 8.0 by
0.1 mol/L NaOH solution.
Further, the ratio of freeze-dried zymoivie in the zymoivte solution is 50-100mg/mt, the mass ratio of trypsin to zymoiyte solution is 0.5%-2%, the two-step enzyme demodulation conditions are pH 7-9, the temperature is 40-60°C, and the enzymoiysis time is 40-80min.
Further, the ratio of freeze-dried zymolyts in the zymoivie solution is 100marme. 1500630 pH is 7.75, the temperature is 53.19°C, and the time is 50min.
Further, the fish scale is from grass cam.
According to the technical scheme, the method has the beneficial effects that fish scales of grass carp, which are byproducts generated from grass camp processing, are used as raw materials, acid decalcification accelerates the dissolution of collagen, and improves the purity of antioxidant peptides, alkaline protsase is firstly used for one-step enzymolysis, and trypsin is used for two-step enzymolysis, so that the content of antioxidant peptides is increased, The method for preparing the high antimudant peptide is simple to operate, non-toxic and harmless, and easy to realize, and the prepared antioxident peptide has higher OH scavenging capacity than dibutylhydroxytoluene (BHT), good oxidation resistance, and high practical value in the fields of health food, medicine, cosmetics and the like.
The following is a detailed description with embodiment Embodiment 1 A preparation method of fish scale gelatin high antioxidant peptide comprises the following specific steps:
1. Pretreatment of raw materials: remove impurities from the surface of grass carp scales, rinse the scales with tap water, and dry them at 40°C for later use, Use C3 moll hydrochloric acid io decaicify fish scales, the ratio of material to liquid of fish scales to hydrochioric acid was 1 10{gmL} and the decalcification time was 1h Rinse the processed scales with tap water for 3-5 times after decalcification, and diving.
2. Preparation of grass carp scale gelating Add the above dried fish scales into distilled water, the ratio of fish scales to water is 115g mlb), the pH is 5.5 (adjusted to the specified pH with hydrochioric acid), and heat and extract the scales in water bath at 80°C for 2 hours. Freeze-drying the extracted solution after suction filtration, 3, One-step enzymoivsis. Dissolve freeze-dried fish phosphorus gelatin with distilled water to obtain fish scale gelatin solution with the mass ratio of 10%. Add alkaline protease for enzymolysis, wherein the amount of alkaline protease is 1% of the weight of fish scale gelatin, and the enzymolysis conditions are as following pH is 80, temperature is 50°C and time is 4h, After enzymolysis, boil for 10 min io inactivate the enzyme, cool the eanzymolysis solution, centrifuge for 10 min at 4000 /min, and freeze 500630 dry the supematant.
4. Two-step enzymoilysis. Dissolve the fresze-dried enzymolysis product in water to prepare a solution with the enzymolysis product ratio of 50 ma/mL, and add trypsin for enzymolysis, wherein the addition amount of trypsin is 0.5%, the pH is 7.8, the temperature is 40°C, and the enzymolysis time is 40 min, AR enzymolyeis, boiling that enzyme for 10 min, centrifuge to obtain supematant, and freeze-drying to obtain the fish scale gelatin high antioxidant peptide, Determination of hydroxyl radical scavenging ability The enzymoiysis products obtained by the above iwo-siep enzymolysis are prepared into 1 mg/ml solution. The 500 ul sample reacts with 150 ul. 8 mM FeS0, 125 pl 20 mid H2O2, 500 LL 3 mM salicylic acid (dissolved in ethanol} in a water bath at 37°C for 20 min and centrifuged for 1 min, The absorbance A, of the supematant was measured at 510 nm, The absorbances of 500 ul distilled water substitute of sample is As, and the absorbance of 125 ul methanol instead of H20Z is A, BHT with the same concentration was used as positive control, and the experiment was repeated three times. OH removal rate is calculated according to the formula: OH clearance ratio(%) | = [1 — a x 100 The experimental results showed that Cs50=0.65 mg/ml is OH clerance ratio of alkaline-trypsin hydrolysate, se 0.78 rmgimi is OH clerance rats of BHT.
Embodiment 2 A preparation method of fish scale gelatin high antioxidant peptide comprises the foliowing specific steps:
1. Pretreatment of raw materials: remove impurities from the surface of grass camp scales, rinse the scales with tap water, and dry them at 40°C for later use, Use 0.3moll hydrochioric acid io decaiciy fish scales, the ratio of material to liquid of fish scales io hydrochloric acid was 1.20{gmL), and the decalcification time was 1h Rinse the processed scales with tap water for 3-5 times after decalcification, and drying.
2. Preparation of grass carp scale gslatin Add the above dried fish scales 500680 distilled water, the ratio of fish scales to water is 1:15(gmL), the pH is 5.5 (adjusted to the specified pH with hydrochioric acid), and heat and extract the scales in water bath at 20°C for 2 hours. Freeze-drying the extracted solution after suction filtration,
3. One-step enzymolysis. Dissolve freeze-dried fish phosphorus gelatin with distilled water to oblain fish scale gelatin solution with the mass ratio of 10%. Add alkaline protease for enzymolysis, wherein the amount of alkaline protease is 1% of the weight of fish scale gelatin, and the enzymolysis conditions are as following: pH is 8.0, temperature is 50°C and time is 4h. After enzymolysis, boil for 10 min to inactivate the enzyme, cool the enzymolysis solution, centrifuge for 10 min at 4000 rimin, and fresze-dry the supernatant.
4. Two-step enzymolysis: Dissolve the freeze-dried enzymolysis product in water to prepare a solution with the enzymoiysis product ratio of 50 mg/mL, and add trypsin for enzymolysis, wherein the addition amount of trypsin is 1%, the pH is 7.8, the temperatura is 50°C, and the enzymolyais time is 50min. AR enzymolysis, boiling that enzyme for 10min, centrifuge to obtain supernatant, and freeze-drying to obtain the fish scale gelatin high antiondant peptide, The OH clesrance ability of fish scale gelatin high antioxidant peptide was determined by the same method as described in Example 1, 1C:=0.65mg/ml is OH clerance ratio of alkaline-trypsin hydrolysate, Ce: 0.78 ma/milis OH clerance rate of BHT.
Embodiment 3 A preparation method of fish scale gelatin high antioxidant peplide comprises the following specific steps:
1. Pretreatment of raw materials: remove impurities from the surface of grass carp scales, rinse the scales with {ap water, and dry them at 40°C for later use, Use 0 7molt hydrochioric acid io decaicify fish scales, the ratio of material io liquid of fish scales to hydrochione acid was 1.30(gmbl), and the decalcification time was 1h Rinse the processed scales with tap water for 3-5 times after decalcification, and driving.
2. Preparation of grass carp scale gelatine Add the above dried fish scales into distilled water, the ratio of fish scales to water is 115g mb), the pH is 5.5 (adjusted to the specified pH with hydrachioric acid}, and heat and extract the scales in water bath 2500630 80°C for 2 hours. Freeze-drying the extracted solution after suction filtration.
3. QOne-step enzymolysis. Dissolve freeze-dried fish phosphorus gelatin with distilled water to obtain fish scale gelatin solution with the mass ratio of 10%. Add alkaline protease for enzymoiysis, wherein the amount of aikaiine protease is 1% of the weight of fish scale gelatin, and the enzymolysis conditions are as following: pH is 8.0, temperature is 50°C and time is 4h, After enzymolysis, boil for 10 min to inactivate the enzyme, cool the enzymolysis solution, centrifuge for 10 min at 4000 r/min, and freeze-dry the supernatant.
4, Two-step enzymolysis. Dissolve the freeze-dried enzymolysis product in water to prepare a solution with the enzymuolysis product ratio of S0mg/mi, and add trypsin for gnzymolysis, wherein the addition amount of trypsin is 2%, the pH is 7.8, the temperature is 60°C, and the enzymolysis time is Sümin, AR enzymolysis, boiling that enzyme for 10min, centrifuge to obtant supernatant, and freeze-drying to obtain the fish scale gelatin high antioxidant peptide.
The OH clearance ability of fish scale gelatin high antioxidant peptide was determined by the same method as described in Example 1, 1Cs=0.68mg/mb is OH clerance ratio of alkaline-trypsin hydrolysate, 10::0.78 ma/miis OH clearance rate of BHT.
Embodiment 4 A preparation method of fish scale gelatin high antioxidant peptide comprises the steps of pretreatment of raw materials, the preparation of grass carp fish scale gelatin and one-step enzymoiysis according to the method described in Embodiment 3. By changing the parameters of the second step enzymolysis, the conditions of the second step enzymolysis of irypsin were optimized. The optimized experimental design is: Three factors, temperature, time and pH, were optimized by response surface methodology. The experimental design scheme and resulls are shown in Table 1.
Embodiment | A (pH) | B(temperature) | C (Time) | weight polypeptide pH temperature)(‘C) | Time (min) | content
16 [ow | sw | wo | sow | 18 800 | ww | wo | wes | [so | sw | wo | 28 | 0 [ow | sw | sw | ssw | 0 [ow | ww | so | sim 2 [00 | ow | wo | sien | Establishment of regression sguation and variance analysis en LU500630 + > ? 5 © + RR 5 = ; “4 8 < © ded gc # SS au > EU» eo = = & & = 8 wo > Pros Ra Es M € Les) = Bb Kr Co = vs = 2 m 8 m 58 SB I” 7 8 5 = PS D {D wo > a” a BR be > Qè a SS En #2 wd {3 2 in va se 2 oe 2 7 5 SS oa SS © 8 og da 5 bu Bony BH ee Lb U Fe =. = ’ Row N wn So Em RS lf m is gs 2 oon ® E ei gi, = 5 = th = bod La Lh — [a — [a Hs — [a Ft Fu ea & 5 = od go © E “8 © d 2 À se ooo a oo > © = NE Em à POS m SZ SS AS SOIT k= = Les Las 1 Je ; La t Sd 3 EC = WS EEE 8 5 2 oe X EP SS S = © 3 & iD _ = Ë, > Sow 8 SS WKN SS < =F kx em =o ia NS à FY » 90 © . NN > 0 RAR RN SD SD = La 5 7 D = & © ce © SC © 89 ee 2 >22 = = bo Ss 558 u Pon oD < © 3 ES SIL E2S 2858 Ë 5 £3 TOR th me = EMS S £3 - SS LA Rs © £ . x % x = SB (E ES; {à Te £3 & tp
Note: * is significant (P < 0.05) and * * is extremely significant (P < 0.01)
From the variance analysis in Table 2, it can be seen thal factor © has a significant affect on the content of low molecular weight peptides in alkaline-trypein hydrolysate (P < 3.01), factors À and B have no significant effect (P > 0.05), and cross-over AB, AC and BC have no significant effect (P > 0,05) According 10 à vaiue, the influence of ihres factors on the content of component IV in the two-step enzymatic hydrolysate is ime > 1500630 temperature > pH, The model established by taking the content of small molecular weight peptide as the response value has a P value of 0.0020, which is extremeiy significant (FF < 0.01). The F-valug and P-value of the unmaiched items were 0.019 and 0.8932, which were not significant (p > 0.05) | shows that both models fit weiß With pH, temperature and time as variables, and the content of low molecular weight peptide in aikaiine-trypsin hydrolysate as response value, regression analysis was carried out by Besign-Expert 80.05, and regression equation (1) was obtained, wherein, Y1 represents the content of low molecular weight peptide.
Y1=-11.338+11.826A+0.560B+0.142C-0.021AB-3.45x10-3AC-2.888x10“BC-
0.671A2-3.633x103B2-9.238x104C2 {1} According to the conditions optimized by response surface methodology, the optimal conditions for the two-step enzymatic hydrolysis of trypsin were that substrats concentration is 100mga/mL, pH/,75, temperature is 53.19°C, time is 50min, and the content of small molecular weight peptide is 53.4088%.
The technical scheme provided by the present invention has been described in detail above. According to the idea of the embodiment of the present invention, there will be some changes in the specific implementation and application scope for the ordinary technicians in the field. To sum up, the contents of this specification should not be construed as limiting the present invention.
Claims (7)
- Claims1. Preparation method of fish scale gelatin high antioxidant peptide is characterized in that if uses fish scales as raw material, and decalcifiss the fish scales, then adopts alkaline protease and trypsin to make two-step enzymatic hydrolysis on fish scales, which obtains the fish scale gelatin peptide with high antioxidant activity.2. According to claim 1, preparation method of fish scale gelatin high antioaidant peptide, is characterized in comprising the following steps: (1) pre-treatment of raw materials. remove impurities on the surface of fish scales, rinse and dry, then decaloify the fish scales with acid solution, rinse and dry the decalcified fish scales, (2} preparation of grass carp scale gelatin: add the dried fish scales into water, heat it for extraction, filter the mixture to obtain extract, and fresze-dry the extract io obtain freeze-dried fish phosphorus gelatin, (3) one-step enzymolysis: dissolve the freeze-dried fish phosphorus gelatin in distilled water to obtain a fish scale gelatin solution, carry out one-step enzymolysis treatment on the fish scale gelatin solution by adopting alkaline proisass, inactivate the enzyme after erzymoilysis, centrifugally separate the enzymolysis solution, and fresze-dry supernatant to obtain freeze-dried enzymolysis products, (4) two-step gnzymolysis: prepare the freeze-dried enzymolysis product into an enzymoiysis product solution, carry OU! two-step enzymolysis with trypsin, inactivate the enzyme after enzymolysis, and centrifuge to take supernatant to obtain the fish scale gelatin high antioxidant peptide.3, Preparation method of fish scale gelatin high antioxidant peptide, according to claim 2, is characterized in that in step 1, the acid solution is 0.30. /moÿL hydrochioric acid, the ratio of fish scales to hydrochloric acid is 1.10~30g:mL, and the decalcification time is 1-3h.À Preparation method of fish scale gelatin high antioxidant peptide, according to claim 2, is characterized in that in step 2, the ratio of material to liquid is 1.15200, the pris 5.0~6.0, the extraction is carried out at 80°C in water bath for more than Z hours, and the pH is adjusted by dripping dilute hydrochloric acid into water.5. Preparation method of fish scale gelatin high antioxidant peplide, according to claim 2, is characterized in that in step 3, the mass fraction of fish phosphorus gelatin in the fish scale gelatin solution is 10%, the addition amount of alkaline protease is 1% 2500630 the mass of fish scale gelatin in the fish scale gelatin solution, the one-siep enzymaolysis conditions are pH 8.0, enzymolysis temperature is 50°C, enzymolysis time is 4h, and pH is adjusted to 8.0 by 4.1 moll NaOH solution,8. Preparation method of fish scale gelatin high antioxidant peptide, according to claim 2, is characterized in thal in step 4, the ratio of freeze-dried zymolvis in the zymolyle solution is 50-100mg/mi, the mass ratio of trypsin to zymolyle solution is 0.5%-2%, the two-step enzyme demodulation conditions are pH 7-8, the temperature is 40-80°C, and the enzymolysis time is 40-B0min.7. Preparation method of fish scale gelatin high antioxidant peptide, according to claim 6, is characterized in that the two-step enzymolysis parameters are as follows: the ratio of freeze-dried zymoivie in the zymoiyte solution is 100mm, the pH is 7.75, the temperature is 53.19°C, and the time is 50min.8. The preparation method of a fish scale gelatin high antioxidant peptide, according to any one of claims 1 to 7, is characterized in that the fish scale is grass camp scale,
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