High-activity collagen peptide and preparation method thereof

Abstract

The invention provides a high-activity collagen peptide and a preparation method thereof, and aims at improving the product yield of the collagen peptide by finely controlling enzymolysis conditions and adopting a specific enzyme activator and effectively controlling the average molecular weight of the collagen peptide so as to enhance the bioavailability and functionality. The preparation method comprises the following steps: S1, crushing fish skin at low temperature, adding the crushed fish skin into an enzyme reaction kettle, adjusting the pH value, and adding composite alkaline protease and an enzyme activator to carry out enzymolysis twice at different temperatures; s2, centrifuging the enzymatic hydrolysate at a high speed, adding activated carbon and citric acid, and filtering to obtain a collagen peptide solution; and S3, carrying out freeze drying on the concentrated solution subjected to ultrafiltration and nanofiltration in the step S3 to obtain the high-activity collagen peptide. The collagen peptide prepared by the method has high DPPH free radical scavenging rate, the molecular weight is lower than 1000Da, the distribution is concentrated, and the product yield and functionality are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen, in particular to a highly active collagen peptide and a preparation method thereof. Background Art

[0002] Collagen peptides have broad application prospects in the food, medicine, and cosmetics industries due to their excellent biological activity and nutritional value. Traditional collagen extraction methods include physical, chemical, and enzymatic methods. Physical methods usually involve high temperature and high pressure, which may cause the loss of active ingredients in collagen peptides; although chemical methods have high extraction efficiency, they may introduce harmful chemicals and affect the safety of the product; and enzymatic methods are considered to be an effective method for extracting collagen peptides due to their mild conditions and high selectivity. Despite this, the existing enzymatic extraction technology still has some shortcomings, such as low extraction efficiency, insufficient retention of active ingredients, and imprecise molecular weight control. These problems limit the application potential and market competitiveness of collagen peptides. Summary of the invention

[0003] In view of the above problems, the present invention provides a highly active collagen peptide and a preparation method thereof.

[0004] To achieve the above objectives, the present invention is implemented by the following technical scheme: a highly active collagen peptide and a preparation method thereof, comprising the following steps: S1: After cleaning the fish skin, pulverize it at low temperature to obtain fine-grained powder, add the fine-grained powder into an enzyme reactor, add purified water, adjust the pH to 7.5-8.2 with a neutral buffer, add a composite alkaline protease and an enzyme activator for enzymolysis, control the temperature at 37° C.-40° C., perform the first enzymolysis for 6-8 hours, obtain the first protease hydrolysate, adjust the pH of the first protease hydrolysate to 6-7 with a neutral buffer, add papain, control the temperature at 55° C.-65° C., perform the second enzymolysis for 8-10 hours, obtain the second protease hydrolysate, and heat the second protease hydrolysate to 80° C. to inactivate the enzyme for 15-20 minutes; S2: The second protease hydrolysate after enzyme inactivation is subjected to high-speed centrifugation at a speed of 3000-4500 rpm for 20-30 min to obtain a centrifugal supernatant, activated carbon and citric acid are added to the centrifugal supernatant, heated to 50°C-60°C, stirred for 20-30 min, allowed to stand for 2-3 h, and filtered to obtain a collagen peptide solution; S3: The collagen peptide liquid is ultrafiltered, and a 3000Da ultrafiltration membrane is selected to retain the collagen peptides. The pressure is maintained at 0.3-0.5 MPa to obtain an ultrafiltration concentrate and an ultrafiltration permeate. The ultrafiltration concentrate is nanofiltered, and a 200Da nanofiltration membrane is selected. The pressure is maintained at 0.6-1.0 MPa to obtain a nanofiltration concentrate. The nanofiltration concentrate is freeze-dried to obtain highly active collagen peptides.

[0005] Preferably, the fineness of the fine-grained powder is 100 mesh.

[0006] Preferably, the composite alkaline protease consists of aminopeptidase and trypsin, and the mass ratio of aminopeptidase to trypsin is 1:3.

[0007] Preferably, the neutral buffer is NaH 2 PO 4 and Na 2 HPO 4 composition.

[0008] Preferably, the enzyme activator consists of theaflavins and catechins, and the mass ratio of theaflavins to catechins is 1:1.

[0009] Preferably, the fish skin, purified water, compound alkaline protease, enzyme activator, papain, activated carbon and citric acid are composed of 100 parts of fine-grained powder, 300-400 parts of purified water, 1-2 parts of compound alkaline protease, 0.6-0.8 parts of enzyme activator, 0.5-1 parts of papain, 2-5 parts of activated carbon and 0.5-1 parts of citric acid in parts by weight.

[0010] Beneficial Effects The present invention provides a highly active collagen peptide and a preparation method thereof. It has the following beneficial effects: 1. The preparation method of the present invention significantly improves the product yield of collagen peptides by finely controlling the enzymatic hydrolysis conditions and using a specific enzyme activator.

[0011] 2. The highly active collagen peptide prepared by the present invention has a higher DPPH free radical scavenging rate.

[0012] 3. The present invention effectively controls the average molecular weight of collagen peptides through precise enzymatic hydrolysis and membrane separation technology. The data in Table 2 show that the average molecular weight of the embodiments is less than 1000 Da and the distribution is relatively concentrated, which helps to improve the bioavailability and functionality of collagen peptides. DETAILED DESCRIPTION

[0013] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0014] Embodiment 1: A method for preparing highly active collagen peptides comprises the following steps: S1: 100 parts of fish skin were cleaned and then crushed at low temperature to obtain fine-grained powder. The fine-grained powder was added to an enzyme reactor, 300 parts of purified water were added, and NaH 2 PO 4 and Na 2 HPO 4 The pH of the first protease solution was adjusted to 6 with a neutral buffer, 0.5 parts of papain were added, the temperature was controlled at 55°C, and the second protease solution was obtained for 8 hours. The second protease solution was heated to 80°C and the enzyme was inactivated for 15 minutes.

[0015] S2: The second protease hydrolysate after enzyme inactivation was subjected to high-speed centrifugation at a centrifugal speed of 3000 rpm for 20 minutes to obtain a centrifugal supernatant. Two parts of activated carbon and 0.5 parts of citric acid were added to the centrifugal supernatant, heated to 50°C, stirred for 20 minutes, allowed to stand for 2 hours, and filtered to obtain a collagen peptide solution.

[0016] S3: The collagen peptide liquid is subjected to ultrafiltration, and a 3000Da ultrafiltration membrane is selected to intercept the collagen peptides, and the pressure is maintained at 0.3MPa to obtain an ultrafiltration concentrate and an ultrafiltration permeate. The concentrate is subjected to nanofiltration, and a 200Da nanofiltration membrane is selected, and the pressure is maintained at 0.6MPa to separate the nanofiltration concentrate and the nanofiltration permeate, and the nanofiltration concentrate is freeze-dried to obtain highly active collagen peptides.

[0017] Embodiment 2: A method for preparing highly active collagen peptides comprises the following steps: S1: 100 parts of fish skin were cleaned and then crushed at low temperature to obtain fine-grained powder. The fine-grained powder was added to an enzyme reactor, 400 parts of purified water were added, and NaH 2 PO 4 and Na 2 HPO 4The pH of the first protease solution was adjusted to 7 with a neutral buffer, 1 part of papain was added, the temperature was controlled at 65°C, and the second protease solution was obtained. The second protease solution was heated to 80°C and the enzyme was inactivated for 20 minutes.

[0018] S2: The second protease hydrolysate after enzyme inactivation was subjected to high-speed centrifugation at a centrifugal speed of 4500 rpm for 30 minutes to obtain a centrifugal supernatant. Five parts of activated carbon and one part of citric acid were added to the centrifugal supernatant, heated to 60°C, stirred for 30 minutes, allowed to stand for 3 hours, and filtered to obtain a collagen peptide solution.

[0019] S3: The collagen peptide liquid is subjected to ultrafiltration, and a 3000Da ultrafiltration membrane is selected to intercept the collagen peptides, and the pressure is maintained at 0.5MPa to obtain an ultrafiltration concentrate and an ultrafiltration permeate. The concentrate is subjected to nanofiltration, and a 200Da nanofiltration membrane is selected, and the pressure is maintained at 1.0MPa, and the nanofiltration concentrate and the nanofiltration permeate are separated, and the nanofiltration concentrate is freeze-dried to obtain highly active collagen peptides.

[0020] Embodiment 3: A method for preparing highly active collagen peptides comprises the following steps: S1: 100 parts of fish skin were cleaned and then crushed at low temperature to obtain fine-grained powder. The fine-grained powder was added to an enzyme reactor, 350 parts of purified water were added, and NaH 2 PO 4 and Na 2 HPO 4 The pH of the first protease solution was adjusted to 6.5 with a neutral buffer, 0.8 parts of papain were added, the temperature was controlled at 60°C, and the second protease solution was obtained for 9 hours. The second protease solution was heated to 80°C and the enzyme was inactivated for 18 minutes.

[0021] S2: The second protease hydrolysate after enzyme inactivation was subjected to high-speed centrifugation at a centrifugal speed of 3750 rpm for 25 minutes to obtain a centrifugal supernatant. 3 parts of activated carbon and 0.8 parts of citric acid were added to the centrifugal supernatant, heated to 55°C, stirred for 25 minutes, allowed to stand for 2.5 hours, and filtered to obtain a collagen peptide solution.

[0022] S3: The collagen peptide liquid is subjected to ultrafiltration, and a 3000Da ultrafiltration membrane is selected to intercept the collagen peptides, and the pressure is maintained at 0.4MPa to obtain an ultrafiltration concentrate and an ultrafiltration permeate. The concentrate is subjected to nanofiltration, and a 200Da nanofiltration membrane is selected, and the pressure is maintained at 0.8MPa to separate the nanofiltration concentrate and the nanofiltration permeate, and the nanofiltration concentrate is freeze-dried to obtain highly active collagen peptides.

[0023] Embodiment 4: A method for preparing highly active collagen peptides comprises the following steps: S1: 100 parts of fish skin were cleaned and then crushed at low temperature to obtain fine-grained powder. The fine-grained powder was added to an enzyme reactor, 320 parts of purified water were added, and NaH 2 PO 4 and Na 2 HPO 4 The pH of the first protease solution was adjusted to 6.8 by using a neutral buffer, 0.7 parts of papain were added, the temperature was controlled at 62°C, and the second protease solution was obtained by heating the second protease solution to 80°C and inactivating the enzyme for 17 minutes.

[0024] S2: The second protease hydrolysate after enzyme inactivation was subjected to high-speed centrifugation at a centrifugal speed of 3500 rpm for 22 minutes to obtain a centrifugal supernatant. 4 parts of activated carbon and 0.6 parts of citric acid were added to the centrifugal supernatant, heated to 52°C, stirred for 22 minutes, allowed to stand for 2.2 hours, and filtered to obtain a collagen peptide solution.

[0025] S3: The collagen peptide liquid is subjected to ultrafiltration, and a 3000Da ultrafiltration membrane is selected to intercept the collagen peptides, and the pressure is maintained at 0.35MPa to obtain an ultrafiltration concentrate and an ultrafiltration permeate. The concentrate is subjected to nanofiltration, and a 200Da nanofiltration membrane is selected, and the pressure is maintained at 0.7MPa, and the nanofiltration concentrate and the nanofiltration permeate are separated, and the nanofiltration concentrate is freeze-dried to obtain highly active collagen peptides.

[0026] Embodiment 5: A method for preparing highly active collagen peptides comprises the following steps: S1: 100 parts of fish skin were cleaned and then crushed at low temperature to obtain fine-grained powder. The fine-grained powder was added to an enzyme reactor, 380 parts of purified water were added, and NaH 2 PO 4 and Na 2 HPO 4The pH of the first protease solution was adjusted to 6.2 using a neutral buffer, 0.6 parts of papain were added, the temperature was controlled at 58°C, and the second protease solution was obtained. The second protease solution was heated to 80°C and the enzyme was inactivated for 16 minutes.

[0027] S2: The second protease hydrolysate after enzyme inactivation was subjected to high-speed centrifugation at a centrifugal speed of 4000 rpm for 28 minutes to obtain a centrifugal supernatant. 3.5 parts of activated carbon and 0.7 parts of citric acid were added to the centrifugal supernatant, heated to 58°C, stirred for 28 minutes, allowed to stand for 2.8 hours, and filtered to obtain a collagen peptide solution.

[0028] S3: The collagen peptide liquid is ultrafiltered, and a 3000Da ultrafiltration membrane is selected to intercept the collagen peptides, and the pressure is maintained at 0.45MPa to obtain an ultrafiltration concentrate and an ultrafiltration permeate. The concentrate is nanofiltered, and a 200Da nanofiltration membrane is selected, and the pressure is maintained at 0.9MPa to separate the nanofiltration concentrate and the nanofiltration permeate, and the nanofiltration concentrate is freeze-dried to obtain highly active collagen peptides.

[0029] Comparative Example 1: A method for preparing highly active collagen peptides comprises the following steps: S1: 100 parts of fish skin are cleaned, cut into pieces and minced to obtain fish skin crumbs. 100 parts of fish skin crumbs are added to an enzyme reactor, 300 parts of purified water are added, and NaH 2 PO 4 and Na 2 HPO 4 The pH of the first protease solution was adjusted to 6 with a neutral buffer, 0.5 parts of papain were added, the temperature was controlled at 55°C, and the second protease solution was obtained for 8 hours. The second protease solution was heated to 80°C and the enzyme was inactivated for 15 minutes.

[0030] S2: The second protease hydrolysate after enzyme inactivation was subjected to high-speed centrifugation at a centrifugal speed of 3000 rpm for 20 minutes to obtain a centrifugal supernatant. Two parts of activated carbon and 0.5 parts of citric acid were added to the centrifugal supernatant, heated to 50°C, stirred for 20 minutes, allowed to stand for 2 hours, and filtered to obtain a collagen peptide solution.

[0031] S3: The collagen peptide liquid is subjected to ultrafiltration, and a 3000Da ultrafiltration membrane is selected to intercept the collagen peptides, and the pressure is maintained at 0.3MPa to obtain an ultrafiltration concentrate and an ultrafiltration permeate. The concentrate is subjected to nanofiltration, and a 200Da nanofiltration membrane is selected, and the pressure is maintained at 0.6MPa to separate the nanofiltration concentrate and the nanofiltration permeate, and the nanofiltration concentrate is freeze-dried to obtain highly active collagen peptides.

[0032] Comparative Example 2: A method for preparing highly active collagen peptides comprises the following steps: S1: 100 parts of fish skin were cleaned and then crushed at low temperature to obtain fine-grained powder. The fine-grained powder was added to an enzyme reactor, 300 parts of purified water were added, and NaH 2 PO 4 and Na 2 HPO 4 The pH of the first protease solution was adjusted to 6 with a neutral buffer, 0.5 parts of papain were added, the temperature was controlled at 55°C, and the second protease solution was obtained. The second protease solution was heated to 80°C and the enzyme was inactivated for 15 minutes.

[0033] S2: The second protease hydrolysate after enzyme inactivation was subjected to high-speed centrifugation at a centrifugal speed of 3000 rpm for 20 minutes to obtain a centrifugal supernatant. Two parts of activated carbon and 0.5 parts of citric acid were added to the centrifugal supernatant, heated to 50°C, stirred for 20 minutes, allowed to stand for 2 hours, and filtered to obtain a collagen peptide solution.

[0034] S3: The collagen peptide liquid is subjected to ultrafiltration, and a 3000Da ultrafiltration membrane is selected to intercept the collagen peptides, and the pressure is maintained at 0.3MPa to obtain an ultrafiltration concentrate and an ultrafiltration permeate. The concentrate is subjected to nanofiltration, and a 200Da nanofiltration membrane is selected, and the pressure is maintained at 0.6MPa to separate the nanofiltration concentrate and the nanofiltration permeate, and the nanofiltration concentrate is freeze-dried to obtain highly active collagen peptides.

[0035] Comparative Example 3: A method for preparing highly active collagen peptides comprises the following steps: S1: 100 parts of fish skin were cleaned and then crushed at low temperature to obtain fine-grained powder. The fine-grained powder was added to an enzyme reactor, 300 parts of purified water were added, and NaH 2 PO 4 and Na 2 HPO 4The pH of the first protease solution was adjusted to 6 with a neutral buffer, 0.5 parts of papain were added, the temperature was controlled at 55°C, and the second protease solution was obtained for 8 hours. The second protease solution was heated to 80°C and the enzyme was inactivated for 15 minutes.

[0036] S2: The second protease hydrolysate after enzyme inactivation was subjected to high-speed centrifugation at a centrifugal speed of 3000 rpm for 20 minutes to obtain a centrifugal supernatant. Two parts of activated carbon were added to the centrifugal supernatant, heated to 50°C, stirred for 20 minutes, allowed to stand for 2 hours, and filtered to obtain a collagen peptide solution.

[0037] S3: The collagen peptide liquid is subjected to ultrafiltration, and a 3000Da ultrafiltration membrane is selected to intercept the collagen peptides, and the pressure is maintained at 0.3MPa to obtain an ultrafiltration concentrate and an ultrafiltration permeate. The concentrate is subjected to nanofiltration, and a 200Da nanofiltration membrane is selected, and the pressure is maintained at 0.6MPa to separate the nanofiltration concentrate and the nanofiltration permeate, and the nanofiltration concentrate is freeze-dried to obtain highly active collagen peptides.

[0038] The fish skin in step S1 in Examples 1 to 5 and Comparative Examples 1 to 3 and the finally prepared highly active collagen peptide were weighed to calculate the product yield, product yield (%) = mass of collagen peptide / mass of fish skin × 100%, the results are shown in Table 1: The high-activity collagen peptides prepared in Examples 1 to 5 and Comparative Examples 1 to 2 were analyzed and tested. The results of the average molecular weight, protein content and DPPH free radical scavenging rate of the high-activity collagen peptides are shown in Table 2: Chromatographic analysis was performed on Examples 1 to 5 and the prepared highly active collagen peptides to determine the percentage of collagen peptides in different molecular weight ranges. The results are shown in Table 3: Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing highly active collagen peptide, characterized in that: The steps include: S1: After cleaning the fish skin, pulverize it at low temperature to obtain fine-grained powder, add the fine-grained powder into an enzyme reactor, add purified water, adjust the pH to 7.5-8.2 with a neutral buffer, add a composite alkaline protease and an enzyme activator for enzymolysis, control the temperature at 37° C.-40° C., perform the first enzymolysis for 6-8 hours, obtain the first protease hydrolysate, adjust the pH of the first protease hydrolysate to 6-7 with a neutral buffer, add papain, control the temperature at 55° C.-65° C., perform the second enzymolysis for 8-10 hours, obtain the second protease hydrolysate, and heat the second protease hydrolysate to 80° C. to inactivate the enzyme for 15-20 minutes; S2: The second protease hydrolysate after enzyme inactivation is subjected to high-speed centrifugation at a speed of 3000-4500 rpm for 20-30 min to obtain a centrifugal supernatant, activated carbon and citric acid are added to the centrifugal supernatant, heated to 50°C-60°C, stirred for 20-30 min, allowed to stand for 2-3 h, and filtered to obtain a collagen peptide solution; S3: The collagen peptide liquid is ultrafiltered, and a 3000Da ultrafiltration membrane is selected to retain the collagen peptides. The pressure is maintained at 0.3-0.5 MPa to obtain an ultrafiltration concentrate and an ultrafiltration permeate. The ultrafiltration concentrate is nanofiltered, and a 200Da nanofiltration membrane is selected. The pressure is maintained at 0.6-1.0 MPa to obtain a nanofiltration concentrate. The nanofiltration concentrate is freeze-dried to obtain highly active collagen peptides.

2. The method for preparing a highly active collagen peptide according to claim 1, characterized in that: The fineness of the fine-grained powder is 100 mesh.

3. The method for preparing a highly active collagen peptide according to claim 1, characterized in that: The composite alkaline protease consists of aminopeptidase and trypsin, and the mass ratio of aminopeptidase to trypsin is 1:

3.

4. The method for preparing a highly active collagen peptide according to claim 1, characterized in that: The neutral buffer consists of NaH2PO4 and Na2HPO4.

5. The method for preparing a highly active collagen peptide according to claim 1, characterized in that: The enzyme activator consists of theaflavins and catechins, and the mass ratio of theaflavins to catechins is 1:

1.

6. The method for preparing a highly active collagen peptide according to claim 1, characterized in that: The fish skin, purified water, compound alkaline protease, enzyme activator, papain, activated carbon and citric acid are 100 parts of fish skin, 300-400 parts of purified water, 1-2 parts of compound alkaline protease, 0.6-0.8 parts of enzyme activator, 0.5-1 parts of papain, 2-5 parts of activated carbon and 0.5-1 parts of citric acid in parts by weight.

7. A highly active collagen peptide, characterized in that: The highly active collagen peptide is prepared according to the method for preparing the highly active collagen peptide according to any one of claims 1 to 6.

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