CN111118094A - Preparation method of cod skin collagen peptide - Google Patents
Preparation method of cod skin collagen peptide Download PDFInfo
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- CN111118094A CN111118094A CN202010111636.9A CN202010111636A CN111118094A CN 111118094 A CN111118094 A CN 111118094A CN 202010111636 A CN202010111636 A CN 202010111636A CN 111118094 A CN111118094 A CN 111118094A
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- collagen peptide
- cod skin
- collagen
- enzymolysis
- mixed material
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- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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Abstract
The invention relates to the technical field of collagen extraction, and particularly relates to a preparation method of cod skin collagen peptide. The method comprises the following steps: mixing the cod skin with water, and extracting at 90-95 ℃ for 2-2.5 hours to obtain a mixed material; adding trypsin for enzymolysis, wherein the mass percent of the added enzyme is 4-5.5%, and the enzymolysis time is 2-3 h; inactivating and centrifuging to obtain a supernatant; decolorizing with activated carbon, removing fishy smell, and filtering with plate frame to obtain decolorized solution; filtering with ultrafiltration membrane, performing ion exchange, concentrating, and freeze drying to obtain cod skin collagen peptide. The extraction rate of the collagen peptide by the method can reach 94-95%. The molecular weight is mainly distributed below 1000Da, the content is as high as more than 80%, the polypeptide mainly comprises a polypeptide mixture of 2-10 amino acids, the contents of hydroxyproline and collagen in the skin of a model mouse can be obviously improved, and the antioxidant activity in the blood of a human body is enhanced, so that the effect of delaying senescence is achieved.
Description
Technical Field
The invention relates to the technical field of collagen extraction, and particularly relates to a preparation method of cod skin collagen peptide.
Background
Collagen is a fibrous protein widely distributed in the skin, hair, bone and blood vessels of animals, and can support and protect the body. The collagen peptide is a polypeptide mixture with the molecular weight less than 3kDa and generated after the collagen is subjected to enzymolysis, and has better absorption rate compared with the collagen; meanwhile, the collagen also has the characteristics which the collagen does not have, such as antioxidant activity, platelet coagulation inhibition activity, anti-tumor activity and the like, and is widely applied to various fields of functional foods, health-care foods, cosmetics and the like at present due to the obvious physiological activity of the collagen.
According to statistics, the annual processing amount of cod in China exceeds 50 ten thousand tons, wherein the cod skin is used as a byproduct and is often discarded, so that environmental pollution and resource waste are caused. Studies show that the cod skin dry matter contains more than 70% of collagen, and is the best raw material for preparing collagen peptide. At present, collagen peptide extracted from cod skin is used as a health food, is developed and applied in the aspects of beautifying and protecting skin, supplementing calcium and strengthening bones, providing immunity and the like, and has less research on the aspects related to anti-aging.
At present, the collagen is generally extracted from the cod skin by a method of hot water cooking and biological enzyme hydrolysis. Let us study on the report that the addition of 5 per mill of papain in the enzymolysis process is most reasonable by detecting the molecular weight distribution of the cod-skin collagen peptide in the process optimization of preparing the collagen peptide from the cod skin published in 2015, and certain help is provided for removing fishy and bitter taste; the addition of the perlite is beneficial to removing the fishy and bitter taste of the cod skin collagen peptide; the sterilization and filtration of the 0.22 mu m microporous filter membrane can filter out microorganisms, and the subsequent sterilization process is simplified; the cod skin collagen peptide yield is not affected after the optimization process. However, the yield of the article is very low, only about 14%.
The aromatic plant discloses the optimal enzymolysis condition in the preparation of cod skin collagen peptide and the protection effect of cod skin collagen peptide on hepatocyte damage: taking trypsin as hydrolase, carrying out enzymolysis at 50 ℃, carrying out pH 6, adding 2000U/g of enzyme, carrying out enzymolysis for 8h, and purifying to obtain the protein peptide GM 2-2-3. A hydrolysis technique using alkaline protease is disclosed in "preparation of cod skin collagen polypeptide and its effect on mouse B16 melanoma cells", which was published in 2006 by Doudantin. After the test of the inventor, the extraction rate of the collagen of the two methods is low.
The method comprises the following steps of cleaning deep sea cod skin for several times to remove impurities and grease, cooking and deoiling at a low temperature of below 80 ℃ to dissolve collagen in the cod skin, degrading the dissolved collagen into micromolecular collagen polypeptide by using biological enzyme, wherein the biological enzyme is at least 2 of papain, bromelain and subtilisin, deodorizing and decolorizing the micromolecular collagen polypeptide by using raw wood active carbon, performing plate-frame filtration on the deodorized and decolorized micromolecular collagen, namely filtering and removing the active carbon by using a biological membrane, separating out the decolorized collagen polypeptide liquid, concentrating the micromolecular collagen polypeptide liquid, performing physical decolorization on the concentrated micromolecular collagen polypeptide liquid, namely, a nanofiltration membrane, a sterilization, a filtration and sterilization, wherein the obtained liquid is prepared by adding a small molecular weight collagen protein which is obtained by soaking and sterilizing the collagen protein in a medium with a relatively high pH value of a collagen, and a relatively high pH value of a collagen protein, and a filtration, and sterilization, the obtained by performing filtration and sterilization, and the obtained by removing the residual active collagen protein from the collagen protein obtained by adding a relatively high pH value of a collagen protein obtained by soaking and a relatively high pH value of a collagen powder, a natural collagen protein which is a natural collagen protein, a natural collagen protein obtained by adding a natural collagen protein which is prepared by performing a relatively high pH value of about 10-6-10-C, and a high-10-one-10-one-10-one-10-one-two-three-two-three-four-three-four-five-.
Disclosure of Invention
In view of the above, the invention provides a preparation method of cod skin collagen peptide. The preparation method can significantly improve the extraction rate of the collagen peptide.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of cod skin collagen peptide, which comprises the following steps:
mixing the cod skin with water according to the weight ratio of 1: (4-6), and extracting at 90-95 ℃ for 2-2.5 h to obtain a mixed material;
adding trypsin into the mixture for enzymolysis, wherein the mass percentage of the trypsin to the mixture is 4-5.5%, and the enzymolysis time is 2-3 hours; inactivating and centrifuging after enzymolysis is finished to obtain supernatant;
adding activated carbon into the supernatant for decolorization and deodorization, and filtering by adopting a plate frame to obtain a decolorized solution;
filtering the decolorized solution by adopting an ultrafiltration membrane, then carrying out ion exchange by adopting an ion exchange column, and then concentrating, freezing and drying to obtain the cod skin collagen peptide.
The invention adopts hot water to extract cod skin collagen, prepares collagen peptide by enzymatic hydrolysis, and obtains the optimal extraction process for preparing the cod skin collagen peptide by a single-factor variable method by taking the extraction rate of the collagen and the collagen peptide as indexes. The anti-aging effect of the cod skin collagen peptide is further researched, a new raw material is provided for research and development of anti-aging related medicines and health-care foods, and a theoretical basis is provided for application of the cod skin collagen peptide.
Preferably, the cod skin is mixed with water in a ratio of 1:4, and extracting for 2 hours at the temperature of 95 ℃.
Preferably, the mass percentage of the trypsin and the mixed material is 4.5%.
Preferably, the enzymolysis time is 2 h.
Preferably, the enzymolysis step also comprises a step of adding an enzyme activity protective agent, wherein the enzyme activity protective agent comprises glycerol, chitotriose and nystose, the mass percent of the glycerol to the mixed material is 0.1-0.3%, the mass percent of the chitotriose to the mixed material is 0.1-0.3%, and the mass percent of the nystose to the mixed material is 0.1-0.3%.
In the specific embodiment provided by the invention, the mass percentage of the glycerol to the mixed material is 0.2%, the mass percentage of the chitotriose to the mixed material is 0.2%, and the mass percentage of the nystose to the mixed material is 0.2%.
Preferably, the inactivation temperature is 90-100 ℃ and the time is 8-12 min.
In the specific embodiment provided by the invention, the inactivation temperature is 98 ℃ and the time is 10 min.
Preferably, the rotation speed of the centrifugation is 3000-5000 rpm, and the time of the centrifugation is 15-25 min.
In the specific embodiment provided by the invention, the rotating speed of the centrifugation is 4000rpm, and the time of the centrifugation is 20 min.
Preferably, the mass percentage of the activated carbon to the supernatant is 1-3%.
In the specific embodiment provided by the invention, the mass percentage of the activated carbon to the supernatant is 2%.
Preferably, the rotating speed of the decoloring and deodorization treatment is 200-400 rpm, and the time is 15-25 min.
In the specific embodiment provided by the invention, the rotating speed of the decoloring and deodorization treatment is 300rpm, and the time is 20 min.
Preferably, the mesh number of the plate-and-frame filtration is 150-200 meshes.
In the specific embodiment provided by the invention, the mesh number of the plate-frame filtration is 180 meshes.
Preferably, the ultrafiltration membrane has a molecular weight cut-off of 1 kDa.
The invention provides a preparation method of cod skin collagen peptide. The preparation method comprises the following steps: mixing the cod skin with water according to the weight ratio of 1: (4-6), and extracting at 90-95 ℃ for 2-2.5 h to obtain a mixed material; adding trypsin into the mixture for enzymolysis, wherein the mass percentage of the trypsin to the mixture is 4-5.5%, and the enzymolysis time is 2-3 hours; inactivating and centrifuging after enzymolysis is finished to obtain supernatant; adding activated carbon into the supernatant for decolorization and deodorization, and filtering by adopting a plate frame to obtain a decolorized solution; filtering the decolorized solution by adopting an ultrafiltration membrane, then carrying out ion exchange by adopting an ion exchange column, and then concentrating, freezing and drying to obtain the cod skin collagen peptide. The invention has the following technical effects:
the experiment of the invention takes the cod skin as the raw material, and the cod skin collagen peptide is prepared by hot water boiling extraction and enzymatic hydrolysis, and the optimal extraction process comprises the following steps: when the material ratio is 1:4, heating at 95 ℃ for 2h, adjusting the reaction condition to pH8.0 and 50 ℃, adding 4-5.5% of trypsin and 0.6% of enzyme activity protective agent, continuing to react for 2h, and freeze-drying after ultrafiltration to obtain the cod skin collagen peptide, wherein the extraction efficiency can reach 94-95%; the molecular weight and the amino acid composition of the obtained cod skin collagen peptide are measured, and experiments show that the polypeptide is mainly distributed below 1000Da, has the content of more than 80 percent and mainly consists of a polypeptide mixture of 2-10 amino acids; the obtained collagen peptide has high content of glycine, proline and glutamic acid, which are respectively 25.38%, 14.83% and 10.56%; while the contents of cystine, tyrosine and histidine were lower, 0.02%, 0.56% and 0.98%, respectively.
Further research on the anti-aging effect of the cod skin collagen peptide shows that: the cod skin collagen peptide can remarkably increase the activity of glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) in the bodies of middle-aged and elderly people, and simultaneously reduce the content of Malondialdehyde (MDA); the content of Hydroxyproline (HYP) and collagen in the skin of a subacute aging model mouse caused by D-galactose can be obviously increased, which indicates that the cod skin collagen peptide can delay the induction of the D-galactose aging process by increasing the activity of antioxidant enzymes in vivo. Therefore, the cod skin collagen peptide has a good development prospect in the aspects of anti-aging related functional foods, health-care products, medicines and the like.
Drawings
FIG. 1 shows the molecular weight distribution of the cod skin collagen peptide measured by HPGPC.
Detailed Description
The invention discloses a preparation method of cod skin collagen peptide, which can be realized by appropriately improving process parameters by the technical personnel in the field by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
In the experiment, cod skin is used as a raw material, cod skin collagen peptide is prepared by hot water boiling extraction and enzymatic hydrolysis, and the optimal extraction process comprises the following steps: when the material ratio is 1:4, heating at 95 ℃ for 2h, adjusting the reaction condition to pH8.0 and 50 ℃, adding 4.5% trypsin and 0.6% enzyme activity protective agent, continuing to react for 2h, and freeze-drying after ultrafiltration to obtain the cod skin collagen peptide, wherein the extraction efficiency can reach 95%; the molecular weight and the amino acid composition of the obtained cod skin collagen peptide are measured, and experiments show that the polypeptide is mainly distributed below 1000Da, has the content of more than 80 percent and mainly consists of a polypeptide mixture of 2-10 amino acids; the obtained collagen peptide has high content of glycine, proline and glutamic acid, which are respectively 25.38%, 14.83% and 10.56%; the content of cystine, tyrosine and histidine is lower, which is respectively 0.02%, 0.56% and 0.98%; further research on the anti-aging effect of the cod skin collagen peptide shows that: the cod skin collagen peptide can remarkably increase the activity of glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) in the bodies of middle-aged and elderly people, and simultaneously reduce the content of Malondialdehyde (MDA); the content of Hydroxyproline (HYP) and collagen in the skin of a subacute aging model mouse caused by D-galactose can be obviously increased, which indicates that the cod skin collagen peptide can delay the induction of the D-galactose aging process by increasing the activity of antioxidant enzymes in vivo. Therefore, the cod skin collagen peptide has a good development prospect in the aspects of anti-aging related functional foods, health-care products, medicines and the like.
The reagents or instruments used in the preparation method of the cod skin collagen peptide provided by the invention can be purchased from the market.
Materials and reagents:
materials: codfish skin was purchased from Weifang North sea Cold storage Co., Ltd; experimental mice (48 female mice of 5 weeks of age, weighing 19-22g) were purchased from the experimental animals Breeding of Jinmengye, Inc.
Reagent: activated charcoal (CAS number 64356-11-3), glycerol (CAS number 56-81-5), chitotriose (CAS number 41708-93-4), nystose (CAS number 13133-07-8) and trypsin (enzyme activity 200U/mg) were purchased from Yunzi Shangcheng-scientific Liaoguan.
The instrument equipment comprises:
50L of electric heating stainless steel reaction kettle (Shandong Long Xinghua chemical and mechanical group); digital display three-purpose electric heating constant temperature water tank water bath (Jincheng Zhijie laboratory instruments factory in the Jintan area); stainless steel constant temperature water tank (jin cheng zhi jie laboratory instruments factory in the gold jar area); BMY60/8000-UB plate and frame filter (Hangzhou Hengsheng filter Co., Ltd.); LG60 model freeze vacuum drying equipment (the same scene new energy science and technology (jiangshan) ltd); 1KDa roll type ultrafiltration membrane (high temperature resistant) (Hangzhou Kaijie membrane separation technology Co., Ltd.)
The invention is further illustrated by the following examples:
example 1 preparation of cod skin collagen peptide
The extraction process for preparing the cod skin collagen peptide comprises the following steps: cod skin-deliming treatment-hot water extraction-enzymolysis-inactivation-decoloration, deodorization-membrane filtration-ion exchange-concentration-drying, and the specific steps are as follows:
1. weighing 100g of cod skin, and extracting with hot water to obtain collagen; the parameters are as follows: the material ratio is 1:4, the extraction temperature is 95 ℃, and the extraction time is 2 h;
2. preparing cod skin collagen peptide by using trypsin for hydrolysis, wherein the enzyme adding amount is 4.5 percent, and the enzymolysis time is 2 hours. In order to improve and protect the activity of trypsin, an enzyme activity protective agent (0.2% of glycerol, 0.2% of chitotriose and 0.2% of sucrose tetrasaccharide) is added for continuous reaction, after the reaction is finished, the protein is inactivated by heating at 98 ℃ for 10min, and the supernatant is obtained by centrifuging at 4000rpm for 20 min.
3. Stirring with 2% active carbon at 300rpm for 20min for decolorizing and deodorizing, and filtering with 180 mesh filter cloth plate-and-frame filter to obtain decolorized solution.
4. The destaining solution is filtered through a 1kDa ultrafiltration membrane.
5. And (3) carrying out ion exchange on the ultrafiltration membrane feed liquid by using an ion exchange column to remove heavy metal ions.
6. And collecting the feed liquid, and concentrating the feed liquid at high temperature by using a material concentration pot to obtain the feed liquid.
7. And (4) freeze-drying the concentrated feed liquid to obtain the cod skin collagen peptide.
Example 2 preparation of cod skin collagen peptide
The enzyme addition was 5%, and the other process parameters were the same as in example 1.
Example 3 preparation of cod skin collagen peptide
The enzyme addition was 5.5%, and the other process parameters were the same as in example 1.
Comparative examples 1 to 19
Comparing the material ratio, the extraction temperature, the extraction time, the enzyme adding amount and the enzymolysis time.
Comparative examples 1 to 4: extracting at 95 ℃ for 2h according to material ratio of 1:2, 1:3, 1:5 and 1:6, collecting supernatant, and detecting content change of cod skin collagen under different material ratio conditions.
Comparative examples 5 to 8: the extraction temperature is set as the gradient of the extraction temperature of the cod skin collagen at 80 ℃, 85 ℃, 90 ℃ and 100 ℃, and after extraction is carried out for 2 hours under the condition that the material ratio is 1:4, the content change of the cod skin collagen under different extraction temperature conditions is detected.
Comparative examples 9 to 12: the extraction time is set as the gradient of the extraction time of the cod skin collagen to be 1h, 1.5h, 2.5h and 3h, and the content change of the cod skin collagen under different extraction time conditions is detected under the conditions that the material ratio is 1:4 and the temperature is 95 ℃.
Comparative examples 13 to 15: the adding amount of the trypsin is set to be 3 percent, 3.5 percent and 4 percent respectively, after hydrolysis is carried out for 2 hours under the conditions of pH8.0 and 50 ℃, the content of the collagen peptide under different enzyme adding amount conditions is detected.
Comparative examples 16 to 19: setting the enzymolysis time as 1h, 3h, 4h and 5h, adjusting the temperature to 50 ℃ at pH8.0, and detecting the content of collagen peptide under different enzymolysis time conditions.
Test example 1 detection of collagen peptide content
In the test example, the content changes of collagen and collagen peptide under different extraction conditions of examples 1 to 3 and comparative examples 1 to 19 are detected by adopting a Kjeldahl method (GB 5009.5-2010). The test results are as follows:
example 1 an extraction process for preparing cod skin collagen peptide is as follows: reacting for 2 hours at the temperature of 95 ℃ in a material ratio of 1:4, adjusting the pH value of the solution to 8.0 by using NaOH, adding 4.5% trypsin, continuing to react for 2 hours at the temperature of 50 ℃, and performing decolorization, deodorization, ultrafiltration and freeze-drying to obtain the cod skin collagen peptide, wherein the yield can reach 95%. In examples 2 and 3, 5% and 5.5% trypsin are added respectively, and the yield reaches 94%.
TABLE 1 Process conditions
| Examples | Material ratio | The extraction temperature is lower | Extraction time h | The amount of enzyme added% | Time of enzymolysis h | The yield is% |
| 1 | 1：4 | 95 | 2 | 4.5 | 2 | 95 |
| 2 | 1：4 | 95 | 2 | 5 | 2 | 94 |
| 3 | 1：4 | 95 | 2 | 5.5 | 2 | 94 |
| 1 | 1：2 | 95 | 2 | 4.5 | 2 | 80 |
| 2 | 1：3 | 95 | 2 | 4.5 | 2 | 87.5 |
| 3 | 1：5 | 95 | 2 | 4.5 | 2 | 92 |
| 4 | 1：6 | 95 | 2 | 4.5 | 2 | 92 |
| 5 | 1：4 | 80 | 2 | 4.5 | 2 | 83 |
| 6 | 1：4 | 85 | 2 | 4.5 | 2 | 80 |
| 7 | 1：4 | 90 | 2 | 4.5 | 2 | 90 |
| 8 | 1：4 | 100 | 2 | 4.5 | 2 | 83.8 |
| 9 | 1：4 | 95 | 1 | 4.5 | 2 | 78.8 |
| 10 | 1：4 | 95 | 1.5 | 4.5 | 2 | 85.5 |
| 11 | 1：4 | 95 | 2.5 | 4.5 | 2 | 92 |
| 12 | 1：4 | 95 | 3 | 4.5 | 2 | 89.7 |
| 13 | 1：4 | 95 | 2 | 3 | 2 | 76.5 |
| 14 | 1：4 | 95 | 2 | 3.5 | 2 | 83 |
| 15 | 1：4 | 95 | 2 | 4 | 2 | 90 |
| 16 | 1：4 | 95 | 2 | 4.5 | 1 | 85 |
| 17 | 1：4 | 95 | 2 | 4.5 | 3 | 90 |
| 18 | 1：4 | 95 | 2 | 4.5 | 4 | 86.8 |
| 19 | 1：4 | 95 | 2 | 4.5 | 5 | 82 |
Test example 2 measurement of molecular weight and amino acid composition
And (3) measuring the molecular weight: weighing 1mg of cod skin collagen peptide, dissolving in distilled water to prepare a solution of 5mg/mL, filtering with a filter membrane, loading on an HPGPC TSK-gel G2500 PWXL chromatographic column at the flow rate of 0.5mL/min, the sample injection volume of 10 mu L, the column temperature of 30 ℃, and the detection wavelength of 220nm, and performing molecular weight determination.
Amino acid component determination: weighing 1mg of cod skin collagen peptide, extracting with hot water, and fixing the volume. Setting the flow rate to be 1.0 ml/min; the sample volume is 5 ml; the column temperature was 38 ℃ and the wavelength was 340nm, and the measurement was carried out by high performance liquid chromatography.
The molecular weight distribution of cod skin collagen peptide extracted under the optimum conditions was measured by HPGPC, and the results of the measurement of collagen extracted in example 1 are shown in fig. 1: the polypeptide produced by enzymolysis is mainly distributed below 1000Da, the content of the polypeptide reaches more than 80 percent, and the polypeptide is a polypeptide mixture mainly containing 2-10 amino acids. Researches show that the short peptide with smaller molecular weight is easier to be absorbed and utilized by human body, so that the obtained cod skin collagen peptide has better application value.
The amino acid composition of the collagen peptides obtained was further examined, and the results are shown in table 2: the collagen peptide is rich in glycine, proline and glutamic acid, and is respectively 25.38%, 14.83% and 10.56%; while the contents of cystine, tyrosine and histidine were lower, 0.02%, 0.56% and 0.98%, respectively. The obtained cod skin collagen peptide has similar amino acid composition with collagen peptide from other sources.
TABLE 2 amino acid composition of cod skin collagen peptide
| Name of amino acid | Content (g/100g) | Name of amino acid | Content (g/100g) |
| Aspartic acid | 5.44 | Isoleucine | 1.47 |
| Cystine | 0.02 | Threonine | 2.74 |
| Glutamic acid | 10.56 | Leucine | 2.58 |
| Valine | 2.51 | Arginine | 5.92 |
| Serine | 3.64 | Lysine | 3.56 |
| Methionine | 1.41 | Alanine | 9.06 |
| Histidine | 0.98 | Proline | 14.83 |
| Phenylalanine | 1.02 | Tyrosine | 0.56 |
| Glycine | 25.38 | Hydroxyproline | 6.37 |
Test example 3 Effect on GSH-PX Activity, SOD Activity, and MDA content in human serum
50 middle aged and elderly people were selected and randomly divided into two groups, an experimental group and a control group, each group consisting of 25 people. The experimental group was administered 3 times a day, 4g each time, 3 times a day for 8 weeks with cod skin collagen peptide prepared in example 1, and the control group was normally fed. 2mL of blood of two groups of experimental objects are respectively taken and rapidly centrifuged at 2-4 ℃ for 30min, then serum is taken, and the activity and content change of the aging related factors are respectively detected strictly according to the operation of GSH-PX (glutathione peroxidase) kit, SOD (total superoxide dismutase) kit and MDA (malondialdehyde) kit instructions. And respectively detecting the changes of the GSH-PX activity, the SOD activity and the MDA content in the serum of the test group and the control group.
As can be seen from Table 3, the activity of GSH-PX and SOD in the test group is obviously increased and the content of MDA is obviously reduced compared with the control group, which shows that the cod skin collagen peptide can improve the activity of GSH-PX and SOD in blood and reduce the content of free radical metabolite MDA.
TABLE 3 influence of cod skin collagen peptide on GSH-PX, SOD and MDA in human serum
Test example 4 Effect on skin HYP and collagen content of aging mice
D-galactose model experimental mice were constructed, and 48 mice were randomly divided into four groups, namely a blank control group, a collagen peptide low dose group, a collagen peptide medium dose group and a collagen peptide high dose group in example 1, and each group had 12 mice. The blank control group is administered with purified water at 10g/kg (body weight), and low, medium and high dosage collagen peptide at 6g/kg, 15g/kg and 30g/kg respectively, and is administered by gastric lavage for 48 days. Taking skin tissue of a mouse, removing skin, removing connective tissue and fat, rinsing with normal saline, wiping with filter paper, and degreasing with acetone and ethyl ether at a ratio of 1: 1. Precisely weighing 50mg, making into 10% skin homogenate, freeze thawing for 3 times, centrifuging at low temperature for 30min, and collecting supernatant to determine HYP content and collagen content.
The content changes of HYP and collagen in the skin of the model mouse and the normal mouse are detected, and the results are shown in the table 4: compared with a control group, the content of HYP and collagen in the skin of a model group mouse is obviously reduced, when low-dose cod collagen peptide and high-dose cod collagen peptide are added respectively, the content of HYP and collagen in the skin of the mouse is obviously increased, and certain dose dependence is presented, which indicates that the cod skin collagen peptide can effectively increase the content of hydroxyproline, so that the collagen synthesis is promoted.
Table 4 effect of cod skin collagen peptides on HYP and collagen in mouse skin
| Group of | HYP content (%) -in the leather garment | Collagen content in skin (%) |
| Normal group | 9.1±0.6 | 65.8±3.5 |
| Model set | 8.4±0.6 | 60.2±3.8 |
| Low dose group | 9.6±0.4 | 68.9±2.7 |
| High dose group | 9.9±0.5 | 71.2±3.9 |
Conclusion
In the experiment, cod skin is used as a raw material, and an optimal extraction process for preparing cod skin collagen peptide is obtained through a series of experiments. The molecular weight is mainly distributed below 1000Da, the content is more than 80 percent, and the polypeptide mixture mainly contains 2-10 amino acids. The amino acid composition has high content of glycine, proline and glutamic acid, and low content of cystine, tyrosine and histidine. The anti-aging activity of the cod skin collagen peptide is further detected, and the result shows that the cod skin collagen peptide can obviously increase the activity of glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) in human blood and reduce the content of Malondialdehyde (MDA); meanwhile, the content of Hydroxyproline (HYP) and collagen in the skin of the D-galactose model mouse can be improved. The result shows that the cod skin collagen peptide has potential application value in the development of anti-aging related medicines and health-care foods.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preparation method of cod skin collagen peptide is characterized by comprising the following steps:
mixing the cod skin with water according to the weight ratio of 1: (4-6), and extracting at 90-95 ℃ for 2-2.5 h to obtain a mixed material;
adding trypsin into the mixed material for enzymolysis, wherein the mass percentage of the trypsin to the mixed material is 4-5.5%, and the enzymolysis time is 2-3 hours; inactivating and centrifuging after enzymolysis is finished to obtain supernatant;
adding activated carbon into the supernatant for decolorization and deodorization, and filtering by adopting a plate frame to obtain a decolorized solution;
filtering the decolorized solution by adopting an ultrafiltration membrane, then carrying out ion exchange by adopting an ion exchange column, and then concentrating, freezing and drying to obtain the cod skin collagen peptide.
2. The method according to claim 1, wherein the cod skin is mixed with water in a ratio of 1:4, and extracting for 2 hours at the temperature of 95 ℃.
3. The method according to claim 1, wherein the mass percentage of the trypsin to the mixed material is 4.5%.
4. The method of claim 1, wherein the enzymolysis time is 2 hours.
5. The preparation method according to claim 1, wherein the step of enzymolysis further comprises a step of adding an enzyme activity protective agent, wherein the enzyme activity protective agent comprises glycerol, chitotriose and nystose, the mass percentage of the glycerol to the mixed material is 0.1-0.3%, the mass percentage of the chitotriose to the mixed material is 0.1-0.3%, and the mass percentage of the nystose to the mixed material is 0.1-0.3%.
6. The method according to claim 1, wherein the inactivation is carried out at a temperature of 90-100 ℃ for 8-12 min.
7. The method according to claim 1, wherein the rotation speed of the centrifugation is 3000-5000 rpm, and the time of the centrifugation is 15-25 min.
8. The method according to claim 1, wherein the mass percentage of the activated carbon to the supernatant is 1% to 3%.
9. The preparation method according to claim 1, wherein the rotating speed of the decoloring and deodorization treatment is 200-400 rpm, and the time is 15-25 min.
10. The preparation method according to any one of claims 1 to 9, wherein the mesh number of the plate-and-frame filtration is 150-200 meshes, and the molecular weight cut-off of the ultrafiltration membrane filtration is 1 kDa.
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| CN111647043B (en) * | 2019-08-07 | 2022-03-22 | 中国农业大学 | Oligopeptide with platelet resisting and antithrombotic functions containing Hyp-Gly sequence |
| CN111574618A (en) * | 2020-06-26 | 2020-08-25 | 润科生物工程（福建）有限公司 | Preparation method of whitening compound protein peptidase hydrolysate, whitening compound protein peptide beverage and preparation method thereof |
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| CN112458139A (en) * | 2020-12-07 | 2021-03-09 | 武汉纺织大学 | Nano active peptide for postpartum repair and production process thereof |
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| CN114214384A (en) * | 2021-12-22 | 2022-03-22 | 海南三元星生物科技股份有限公司 | Marine organism-derived collagen peptide, and extraction method and application thereof |
| CN114983857A (en) * | 2022-05-25 | 2022-09-02 | 杭州铂赛生物科技有限公司 | Preparation method and application of pacific codfish skin gelatin peptide microneedle |
| CN114983857B (en) * | 2022-05-25 | 2023-09-01 | 杭州铂赛生物科技有限公司 | Preparation method and application of Pacific cod skin gelatin peptide microneedle |
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