CN116284342A - Antioxidant collagen peptide prepared by enzyme method and preparation method thereof - Google Patents
Antioxidant collagen peptide prepared by enzyme method and preparation method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- 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
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Abstract
The invention provides an antioxidant collagen peptide prepared by an enzymatic method and a preparation method thereof, and relates to the technical field of biological medicine, wherein the raw material of the antioxidant collagen peptide is pigskin, and the enzymolysis process of the antioxidant collagen peptide comprises the following two steps: preliminary enzymolysis and secondary enzymolysis, wherein the preliminary enzymolysis uses neutral protease for enzymolysis, and the preliminary enzymolysis uses neutral protease; after the preliminary enzymolysis is completed, the pH value of an enzymolysis solution is adjusted: adjusting the enzymolysis solution to be slightly acidic, and performing secondary enzymolysis by using acid protease; or adjusting the enzymolysis solution to be weak alkaline, and performing secondary enzymolysis by using alkaline protease. In the enzymolysis process, through carrying out preliminary enzymolysis and secondary enzymolysis to the raw materials, the full going on in the enzymolysis process is guaranteed to reduce the pigskin of incomplete enzymolysis after the enzymolysis, thereby improved the raw materials utilization ratio in the production process, reduced manufacturing cost.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to an enzyme method for preparing antioxidant collagen peptide and a preparation method thereof.
Background
The collagen peptide is produced by taking fresh animal tissues (including skin, bones, tendons, scales and the like) rich in collagen as raw materials through extraction, hydrolysis and refining, and the relative molecular mass of the produced collagen peptide is lower than 10000. In the prior art, most of collagen peptide is produced by taking fish as a raw material, and collagen peptide produced by taking pigskin as a raw material is rare; however, since pork consumption in the market is much greater than that in fish, the raw material obtaining method is simpler, and secondly, since human and animals such as pigs, cows and sheep are highly homologous in terms of biological homology, they are all higher mammals, and therefore, the degree of similarity between pig collagen and human collagen is relatively high, and thus, the pig collagen and the human collagen are easily absorbed by human bodies.
The invention discloses a method for extracting active collagen peptide from pigskin, which mainly comprises the steps of cutting pigskin, washing, carrying out enzymolysis, filtering, inactivating enzyme, filtering, concentrating and drying to obtain the active collagen peptide, wherein the specific enzymolysis process comprises the following steps: the temperature and the pH value of the whole slurry are regulated, then compound protease with the weight of 2-5 per mill of the raw material pigskin is added for reaction for 4-6 hours, so that the aim of enzymolysis is fulfilled.
Disclosure of Invention
The invention aims to solve the technical problems that in the prior art, a large amount of non-enzymatic pigskin is easy to generate when the pigskin is subjected to enzymatic hydrolysis, so that the cost is increased, the secondary utilization rate of the residual pigskin is lower, and the treatment is inconvenient.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
an enzyme method for preparing antioxidant collagen peptide, wherein the raw material of the collagen peptide is pigskin, and the enzymolysis process of the collagen peptide comprises the following two steps: primary enzymolysis and secondary enzymolysis, wherein the primary enzymolysis uses neutral protease for enzymolysis, and the secondary enzymolysis uses acid protease or alkaline protease for enzymolysis.
Preferably, the clearance of the collagen peptide to DPPH is greater than 80%; the clearance rate of the collagen peptide to the hydroxyl radical is 50-60%.
The application also provides a preparation method for preparing the antioxidant collagen peptide by an enzymatic method, which is used for preparing the antioxidant collagen peptide, wherein the enzymolysis process is divided into two steps, and neutral protease is used for primary enzymolysis; after the preliminary enzymolysis is completed, the pH value of an enzymolysis solution is adjusted: adjusting the enzymolysis solution to 5.5-6.5, and performing secondary enzymolysis by using acid protease; or adjusting the enzymolysis solution to 7.5-8.5, and performing secondary enzymolysis by using alkaline protease.
Preferably, after the primary enzymolysis is finished, 0.2% of edible sodium carbonate is used for adjusting the pH value of the enzymolysis solution to 7.5, the addition amount of alkaline protease is 1-5% of the mass of the pigskin in the secondary enzymolysis process, the secondary enzymolysis time is 7-9h, and a constant-temperature water bath kettle is used for keeping the temperature between 40 ℃ and 48 ℃ in the enzymolysis process.
Preferably, after the primary enzymolysis is finished, the pH value of the enzymolysis is adjusted to 6.5 by using 0.35% of edible acetic acid, and in the secondary enzymolysis process, the addition amount of the acid protease is 2-10% of the mass of the pigskin, and the secondary enzymolysis time is 8-10h. In the enzymolysis process, a constant-temperature water bath kettle is used for keeping the temperature between 35 and 40 ℃.
Preferably, in the primary enzymolysis process, the temperature of the enzymolysis solution is kept between 40 and 50 ℃ by using a constant water bath kettle, and the enzymolysis time is 3 to 6 hours.
Preferably, in the enzymolysis process, a stirring rod is used for stirring the constant-temperature water bath and the enzymolysis solution, wherein the stirring speed in the constant-temperature water bath is 20-40rpm, and the stirring speed in the enzymolysis solution is 100-150rpm.
Preferably, the preparation method comprises the following specific steps:
s1: selecting pigskin, cleaning the pigskin, removing hair roots and fat on the pigskin, and cutting the pigskin into pieces;
s2: removing fat on the surface of the pigskin by using an isopropanol solution;
s3: removing the foreign proteins in the pigskin by using NaCl solution;
s4: taking out the dried pigskin in the step S3, putting the pigskin into distilled water, heating the distilled water to 100 ℃, and refluxing and steaming for 1h;
s5: performing enzymolysis;
s6: placing the mixed solution subjected to enzymolysis in the step S5 into a constant-temperature water bath kettle, keeping the temperature between 90 and 100 ℃, and standing for 10 to 20 minutes to achieve the purpose of enzyme inactivation;
s7: cooling the mixture obtained in the step S6, and centrifuging for 20min to separate a fat layer from the pigskin which is not subjected to enzymolysis;
s8: extracting the supernatant liquid after centrifugal separation, and carrying out vacuum suction filtration to obtain crude extracted collagen;
s9: and (3) evaporating and concentrating: the solution after vacuum filtration was compressed using a rotary evaporator,
s10: collagen peptide powder was obtained using a spray drying tower in combination with a granulator.
Preferably, in the step S1, the pigskin is washed by using a solution of 0.9% NaCl to remove impurities on the surface of the pigskin.
Preferably, the concentration of the isopropanol solution in the S2 is 10%, and the mass ratio of the isopropanol solution to the pigskin is 20:1, the treatment environment temperature is 4 ℃, and the treatment time is 20-25h.
The application also provides an antioxidant collagen peptide which is prepared by using the preparation method.
According to the preparation method of the antioxidant collagen peptide by the enzymatic method, in the enzymolysis process, the raw materials are subjected to primary enzymolysis and secondary enzymolysis, so that the full progress in the enzymolysis process is ensured, the production of pigskin which is not subjected to enzymolysis after enzymolysis is reduced, the utilization rate of the raw materials in the production process is improved, and the production cost is reduced.
Drawings
FIG. 1 is a graph showing the comparison of DPPH scavenging ability of antioxidant collagen according to one embodiment of the present invention;
FIG. 2 is a graph showing the comparison of the scavenging ability of antioxidant collagen to hydroxyl radicals according to one embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
The application provides a preparation method for preparing antioxidant collagen peptide by an enzymatic method, which comprises the following steps:
s1: raw material preparation
In one embodiment, the raw materials are pigskin, fresh pigskin with transparent cortex is selected, and the pigskin is cleaned by using 0.9% NaCl solution to remove impurities on the surface of the pigskin;
placing the pigskin after cleaning in a ventilation place for airing, and then removing the hair roots and fat on the pigskin; the pigskin is then cut into small pieces, in one embodiment the pigskin pieces have a maximum dimension of 3mm or less.
S2: degreasing:
completely immersing the pigskin treated in the step S1 in an isopropanol solution, wherein the concentration of the isopropanol solution is 10%, and the mass ratio of the isopropanol solution to the pigskin is 20:1, treating at the temperature of 4 ℃ for 20-25 hours to remove the surface fat of the pigskin;
after the soaking is completed, the pigskin is removed and rinsed with distilled water, which in one embodiment is maintained at 40 ℃. The flushing times are 2-5 times.
S3: removing the hybrid protein:
in one embodiment, the washed pigskin is immersed in a 2% NaCl solution, the mass ratio of the pigskin to the NaCl solution is 1:9, and the pigskin is kept at normal temperature for 6-10 hours, so as to remove the foreign proteins in the pigskin. After the soaking is completed, the pigskin is taken out and dried in a ventilation environment, and the environmental temperature is kept at 3 ℃ in the drying process.
S4: reflux cooking:
taking out the pig skin aired in the step S3, putting the pig skin into distilled water, and in one embodiment, heating the distilled water to 100 ℃ and refluxing and steaming for 1h, wherein the mass ratio of the pig skin to the distilled water is 1:7.
S5: enzymolysis:
standing and cooling distilled water steamed in the step S4 to 55 ℃, then adding one or two of papain and flavourzyme for enzymolysis, and keeping the temperature between 40 and 50 ℃ by using a constant-temperature water bath kettle in the enzymolysis process for 3 to 6 hours;
after the preliminary enzymolysis is completed, an adjusting liquid is added to adjust the pH value of the enzymolysis solution to 7.5, and in one embodiment, the adjusting liquid is 0.2% edible alkali.
Then adding alkaline protease with the mass of 1-5% of that of pigskin for enzymolysis for 7-9h. In the enzymolysis process, a constant-temperature water bath kettle is used for keeping the temperature between 40 and 48 ℃.
In another embodiment, after the preliminary enzymolysis is completed, the pH value of the enzymolysis solution is adjusted to 6.5 by using an adjusting liquid, wherein the adjusting liquid is 0.35% of edible acetic acid. Adding acid protease accounting for 2% -10% of the pigskin mass for enzymolysis for 8-10h. In the enzymolysis process, a constant-temperature water bath kettle is used for keeping the temperature between 35 and 40 ℃.
And in the enzymolysis process, stirring the constant-temperature water bath and the enzymolysis solution by using stirring rods, wherein the stirring speed in the constant-temperature water bath is 20-40rpm, and the stirring speed in the enzymolysis solution is 100-150rpm. Stirring the constant-temperature water bath through the stirring rod, so that a container of the enzymolysis solution in the constant-temperature water bath can be in a vibrating state, and stirring the enzymolysis solution through the stirring rod, thereby ensuring that protease and pigskin are fully contacted.
S6: enzyme inactivation:
and (3) placing the solution subjected to enzymolysis in a constant-temperature water bath kettle, keeping the temperature between 90 and 100 ℃, and standing for 10 to 20 minutes to achieve the aim of enzyme inactivation.
S7: and (3) centrifugal separation:
and (3) standing the solution subjected to enzyme inactivation in the step (S5) in a dust-free environment, cooling to normal temperature, and centrifuging for 20min to separate a fat layer from the pigskin which is not subjected to enzymolysis.
S8: vacuum suction filtration:
taking out the centrifuged supernatant, and then carrying out vacuum suction filtration to obtain the crude extracted collagen peptide.
S9: and (3) evaporating and concentrating:
compressing the solution by using a rotary evaporator, wherein the temperature is kept at 50 ℃ in the compression process, so as to obtain pigskin collagen peptide concentrated solution, and the obtained pigskin collagen peptide concentrated solution is 1/3 of the original volume.
S10: and (3) drying and granulating:
the collagen peptide powder was obtained by combining a spray drying tower with a granulator.
The application also provides an enzyme method for preparing the antioxidant collagen peptide, wherein the antioxidant collagen peptide is collagen peptide powder prepared by the preparation method, the amino acid sequence of the collagen peptide is Gly-X-Pro, gly is glycine, pro is proline, and X is any amino acid except glycine and proline
The present application is illustrated below in connection with specific embodiments:
example 1:
based on the preparation method for preparing the antioxidant collagen peptide by the enzymatic method, 500g of fresh pigskin with transparent cortex is selected, and the pigskin is cleaned by using a solution of 0.9% NaCl to remove impurities on the surface of the pigskin;
placing the cleaned pigskin at a ventilation place for airing, removing visible hair roots and fat on the surface of the pigskin after airing, and then cutting the pigskin into small pieces with the diameter of 3mm or 3mm, wherein in one embodiment, a mincing device is used for mincing the pigskin;
immersing the treated pigskin in isopropanol solution with concentration of 10% and mass of 10% 4 And g, keeping the ambient temperature at 4 ℃ to remove invisible fat on the surface of the pigskin.
After the treatment time is 20 hours, the pigskin is taken out and washed by distilled water for 4 times at 40 ℃;
immersing the rinsed pigskin in 4.5 x 10 3 g of NaCl solution, wherein the concentration of the NaCl solution is 2%, the normal temperature is kept in the soaking process, the pigskin is taken out after being soaked for 7 hours, the pigskin is dried in a ventilation environment, and the environment temperature is kept at 3 ℃ in the drying process.
Placing the dried pigskin into a container with a volume of 3.5 x 10 3 g of distilled water, heating the distilled water to 100 ℃, and then refluxing and steaming for 1h.
After the reflux stewing is finished, stewing distilled water is firstly kept still and cooled to 55 ℃, then papain is added for enzymolysis, a constant-temperature water bath is used for keeping the temperature between 40 and 50 ℃ in the enzymolysis process, and the enzymolysis time is 6 hours;
after the preliminary enzymolysis is finished, 0.2 percent of edible flour alkali is added, and the pH value of distilled water is adjusted to 7.5; after the adjustment is finished, adding alkaline protease accounting for 2% of the pigskin mass for enzymolysis for 9 hours, and using a constant-temperature water bath kettle in the enzymolysis process to keep the enzymolysis temperature between 40 ℃ and 45 ℃.
In the enzymolysis process, stirring the constant-temperature water bath and the enzymolysis solution respectively by using stirring rods, wherein in the preliminary enzymolysis and the alkaline protease enzymolysis process, the stirring speed in the constant-temperature water bath is 20rpm, and the stirring speed of the enzymolysis solution is as follows: the stirring speed of the primary enzymolysis solution is 150rpm, and the stirring speed of the stirring rod in the enzymolysis process of alkaline protease is 120rpm.
After the enzymolysis is completed, the temperature of the constant-temperature water bath kettle is increased to 95 ℃, and the constant-temperature water bath kettle stands for 15min to inactivate enzymes.
And then carrying out centrifugal separation, vacuum suction filtration, evaporation concentration, drying and granulation on the enzymatic hydrolysis solution subjected to enzyme inactivation, thereby obtaining collagen peptide powder A.
Example 2:
based on the preparation method for preparing the antioxidant collagen peptide by the enzymatic method, 500g of fresh pigskin with transparent cortex is selected, and the pigskin is cleaned by using a solution of 0.9% NaCl to remove impurities on the surface of the pigskin;
placing the cleaned pigskin at a ventilation position for airing, removing visible hair roots and fat on the surface of the pigskin after airing, and then cutting the pigskin into 3mm x 3mm small pieces;
immersing the treated pigskin in isopropanol solution with concentration of 10% and mass of 10% 4 And g, keeping the ambient temperature at 4 ℃ to remove invisible fat on the surface of the pigskin.
After the treatment time is 22 hours, the pigskin is taken out and washed by distilled water for 5 times at 40 ℃;
immersing the rinsed pigskin in 4.5 x 10 3 g of NaCl solution, wherein the concentration of the NaCl solution is 2%, the normal temperature is kept in the soaking process, the pigskin is taken out after being soaked for 9 hours, the pigskin is dried in a ventilation environment, and the environment temperature is kept at 3 ℃ in the drying process.
Placing the dried pigskin into a container with a volume of 3.5 x 10 3 g of distilled water, heating the distilled water to 100 ℃, and then refluxing and steaming for 1h.
After the reflux stewing is finished, stewing distilled water is firstly kept still and cooled to 55 ℃, then flavourzyme is added for enzymolysis, a constant-temperature water bath is used for keeping the temperature between 40 ℃ and 50 ℃ in the enzymolysis process, and the enzymolysis time is 5 hours;
after the preliminary enzymolysis is finished, 0.35 percent of edible acetic acid is added, and the pH value of distilled water is adjusted to 6.5; after the adjustment is finished, pepsin accounting for 2% of the pigskin mass is added for enzymolysis for 8 hours, and a constant-temperature water bath kettle is used in the enzymolysis process, so that the enzymolysis temperature is kept between 30 ℃ and 40 ℃.
In the enzymolysis process, stirring the constant-temperature water bath and the enzymolysis solution respectively by using a stirring rod, wherein in the preliminary enzymolysis process, the stirring speed in the constant-temperature water bath is 40rpm, the stirring speed in the enzymolysis solution is 130rpm, and in the enzymolysis process by using acid protease, the stirring speed of the stirring rod in the constant-temperature water bath is 35rpm; the stirring speed of the primary enzymolysis solution is 150rpm, and the stirring speed of a stirring rod in the enzymolysis solution in the enzymolysis process of the acid protease is 100rpm.
After the enzymolysis is completed, the temperature of the constant-temperature water bath kettle is increased to 100 ℃, and the constant-temperature water bath kettle stands for 20min to inactivate enzymes.
And then carrying out centrifugal separation, vacuum suction filtration, evaporation concentration, drying and granulation on the enzymatic hydrolysis solution subjected to enzyme inactivation, thereby obtaining collagen peptide powder B.
Example 3
Based on the preparation method for preparing the antioxidant collagen peptide by the enzymatic method, 500g of fresh pigskin with transparent cortex is selected, and the pigskin is cleaned by using a solution of 0.9% NaCl to remove impurities on the surface of the pigskin;
placing the cleaned pigskin at a ventilation position for airing, removing visible hair roots and fat on the surface of the pigskin after airing, and then cutting the pigskin into 3mm x 3mm small pieces;
immersing the treated pigskin in isopropanol solution with concentration of 10% and mass of 10% 4 And g, keeping the ambient temperature at 4 ℃ to remove invisible fat on the surface of the pigskin.
After the treatment time is 20 hours, the pigskin is taken out and washed by distilled water for 4 times at 40 ℃;
immersing the rinsed pigskin in 4.5 x 10 3 g of NaCl solution, wherein the concentration of the NaCl solution is 2%, the normal temperature is kept in the soaking process, the pigskin is taken out after being soaked for 10 hours, the pigskin is dried in a ventilation environment, and the environment temperature is kept at 3 ℃ in the drying process.
Placing the dried pigskin into a container with a volume of 3.5 x 10 3 g of distilled water, heating the distilled water to100℃and then reflux-steaming for 1h.
After the reflux stewing is finished, stewing distilled water is firstly kept stand and cooled to 55 ℃, then papain and flavourzyme mixture is added for enzymolysis, and a constant-temperature water bath kettle is used for keeping the temperature between 40 ℃ and 50 ℃ in the enzymolysis process, so that the enzymolysis time is 3 hours;
after the preliminary enzymolysis is finished, 0.35 percent of edible acetic acid is added, and the pH value of distilled water is adjusted to 6.5; after the adjustment is finished, trypsin and chymotrypsin accounting for 2% of the pigskin mass are added for enzymolysis, the mass ratio of the trypsin to the chymotrypsin is 1:1, the enzymolysis time is 8 hours, and a constant-temperature water bath kettle is used in the enzymolysis process, and the enzymolysis temperature is kept between 30 ℃ and 40 ℃.
In the enzymolysis process, stirring the constant-temperature water bath and the enzymolysis solution respectively by using a stirring rod, wherein in the preliminary enzymolysis process, the stirring speed in the constant-temperature water bath is 20rpm, the stirring speed in the enzymolysis solution is 150rpm, and in the enzymolysis process by using acid protease, the stirring speed of the stirring rod in the constant-temperature water bath is 40rpm; the stirring speed of the primary enzymolysis solution is 100rpm, and the stirring speed of a stirring rod in the enzymolysis solution in the enzymolysis process of the acid protease is 100rpm.
After the enzymolysis is completed, the temperature of the constant-temperature water bath kettle is increased to 100 ℃, and the constant-temperature water bath kettle stands for 20min to inactivate enzymes.
And then carrying out centrifugal separation, vacuum suction filtration, evaporation concentration, drying and granulation on the enzymatic hydrolysis solution subjected to enzyme inactivation, thereby obtaining collagen peptide powder C.
The DPPH scavenging ability and the hydroxyl radical scavenging ability were measured in this application using the collagen peptide powders A, B, C obtained in examples 1 to 3, respectively, to demonstrate the antioxidant activity of the antioxidant collagen peptides prepared using the preparation method of this application:
specifically, example 4 and example 5.
Example 4:
antioxidant Activity test 1 (DPPH method):
five groups of DPPH-ethanol solutions with the concentration of 125 mu mol/L are prepared, four groups of DPPH-ethanol solutions are respectively mixed with collagen peptide powder A, B, C and deionized water in the examples 1-3, and the other group is taken as a blank experiment group, and the blank experiment group is kept away from light for 30min after violent shaking;
the absorbance was then measured at 517nm, wherein the absorbance of the collagen peptide powder was designated A i (A A 、A B 、A C ) The absorbance of the deionized water group was designated A 0 Absorbance of the blank group was recorded as a E The clearance of DPPH free radical from each sample was calculated according to the following formula, repeated 3-6 times in parallel, and then averaged.
Referring to fig. 1, it can be seen from fig. 1 that the clearance of the antioxidant collagen peptide prepared by the preparation method of the present application to DPPH is more than 80%.
Example 5:
judgment of the hydroxyl radical scavenging ability:
reagent preparation: phosphate buffer solution with the pH value of 7.4 and the mol/L of 0.2;
1.865mmol/L phenanthroline-absolute ethanol solution;
FeSO of 1.865mmol/L 4 ·7H 2 An O solution;
ascorbic acid;
0.03% (v/v) H 2 O 2 。
The experiments were divided into five groups (collagen peptide group, control group and injury group): specifically, 1ml of 1.865mmol/L phenanthroline-absolute ethanol solution is placed in a test tube with a stopper, then phosphate buffer solution (2 ml) with the concentration of 0.2mol/L and the pH value of 7.4 and 1ml of sample are respectively added, and after being fully and evenly mixed, 1ml of 1.865mmol/L FeSO is added 4 ·7H 2 O solution, mixing again, adding 0.03% (v/v) H 2 O 2 The absorbance A of each group of mixed solution was measured at 536nm after maintaining the temperature at 37℃for 60min using a constant temperature water bath S (A A 、A B 、A C ) Measurement of absorbance values Using distilled Water instead of sample as blank groupIs A 0 Distilled water is used for replacing H 2 O 2 As an injury group, its absorbance value A was measured n The hydroxyl radical scavenging rate of the collagen peptide was calculated by using ascorbic acid as a positive control instead of the sample according to the following formula:
referring to fig. 2, it can be seen from fig. 2 that the hydroxy radical clearance of the antioxidant collagen peptide prepared according to the preparation method of the present application is about 55%.
According to the preparation method for preparing the antioxidant collagen peptide by the enzymatic method, in the enzymolysis process, the raw materials are subjected to primary enzymolysis and secondary enzymolysis, so that the full progress in the enzymolysis process is ensured, the pigskin which is subjected to enzymolysis and is not completed is reduced, the raw material utilization rate in the production process is improved, and the production cost is reduced.
Claims (10)
1. An enzyme method for preparing antioxidant collagen peptide, which is characterized in that: the collagen peptide raw material is pigskin, and the enzymolysis process of the collagen peptide is divided into two steps: primary enzymolysis and secondary enzymolysis, wherein the primary enzymolysis uses neutral protease for enzymolysis, and the secondary enzymolysis uses acid protease or alkaline protease for enzymolysis.
2. The method for preparing the antioxidant collagen peptide by using the enzyme method according to claim 1, wherein the method comprises the following steps of: the clearance rate of the collagen peptide to DPPH is more than 80%; the clearance rate of the collagen peptide to the hydroxyl radical is 50-60%.
3. A method for preparing an antioxidant collagen peptide by an enzymatic method, which is used for preparing the antioxidant collagen peptide according to claim 1 or 2, and is characterized in that: the enzymolysis process is divided into two steps, wherein neutral protease is used for preliminary enzymolysis; after the preliminary enzymolysis is completed, the pH value of an enzymolysis solution is adjusted: adjusting the enzymolysis solution to 5.5-6.5, and performing secondary enzymolysis by using acid protease; or adjusting the enzymolysis solution to 7.5-8.5, and performing secondary enzymolysis by using alkaline protease.
4. The method for preparing the antioxidant collagen peptide by using the enzymatic method according to claim 3, wherein the method comprises the following steps: after the primary enzymolysis is finished, the pH value of the enzymolysis solution is adjusted to 7.5 by using 0.2% edible sodium carbonate, the adding amount of alkaline protease is 1-5% of the mass of the pigskin in the secondary enzymolysis process, the secondary enzymolysis time is 7-9h, and the temperature is kept between 40-48 ℃ by using a constant-temperature water bath kettle in the enzymolysis process.
5. The method for preparing the antioxidant collagen peptide by using the enzymatic method according to claim 3, wherein the method comprises the following steps: after the primary enzymolysis is finished, edible acetic acid with the concentration of 0.35% is used for leading the pH value of the enzymolysis to be 6.5, and in the secondary enzymolysis process, the addition amount of the acid protease is 2-10% of the mass of the pigskin, and the secondary enzymolysis time is 8-10h. In the enzymolysis process, a constant-temperature water bath kettle is used for keeping the temperature between 35 and 40 ℃.
6. The method for preparing the antioxidant collagen peptide by using the enzymatic method according to claim 3, wherein the method comprises the following steps: in the primary enzymolysis process, the temperature of the enzymolysis solution is kept between 40 ℃ and 50 ℃ by using a constant-temperature water bath kettle, and the enzymolysis time is 3-6h.
7. The method for preparing the antioxidant collagen peptide by the enzymatic method according to claim 6, wherein the method comprises the following steps: in the enzymolysis process, stirring the constant-temperature water bath kettle and the enzymolysis solution by using a stirring rod, wherein the stirring speed in the constant-temperature water bath kettle is 20-40rpm, and the stirring speed in the enzymolysis solution is 100-150rpm.
8. The method for preparing the antioxidant collagen peptide by using the enzymatic method according to claim 3, wherein the method comprises the following steps: the preparation method comprises the following specific steps:
s1: selecting pigskin, cleaning the pigskin, removing hair roots and fat on the pigskin, and cutting the pigskin into pieces;
s2: removing fat on the surface of the pigskin by using an isopropanol solution;
s3: removing the foreign proteins in the pigskin by using NaCl solution;
s4: taking out the dried pigskin in the step S3, putting the pigskin into distilled water, heating the distilled water to 100 ℃, and refluxing and steaming for 1h;
s5: performing enzymolysis;
s6: placing the mixed solution subjected to enzymolysis in the step S5 into a constant-temperature water bath kettle, keeping the temperature between 90 and 100 ℃, and standing for 10 to 20 minutes to achieve the purpose of enzyme inactivation;
s7: cooling the mixture obtained in the step S6, and centrifuging for 20min to separate a fat layer from the pigskin which is not subjected to enzymolysis;
s8: extracting the supernatant liquid after centrifugal separation, and carrying out vacuum suction filtration to obtain crude extracted collagen;
s9: and (3) evaporating and concentrating: the solution after vacuum filtration was compressed using a rotary evaporator,
s10: collagen peptide powder was obtained using a spray drying tower in combination with a granulator.
9. The method for preparing the antioxidant collagen peptide by the enzymatic method according to claim 8, wherein the method comprises the following steps: in the step S1, the pigskin is washed by using a solution of 0.9% NaCl, so that impurities on the surface of the pigskin are removed.
10. The method for preparing the antioxidant collagen peptide by the enzymatic method according to claim 8, wherein the method comprises the following steps: the concentration of the isopropanol solution in the S2 is 10%, and the mass ratio of the isopropanol solution to the pigskin is 20:1, the treatment environment temperature is 4 ℃, and the treatment time is 20-25h.
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