CN108690131B - Kangaroo skin collagen peptide and application thereof - Google Patents
Kangaroo skin collagen peptide and application thereof Download PDFInfo
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- CN108690131B CN108690131B CN201810558302.9A CN201810558302A CN108690131B CN 108690131 B CN108690131 B CN 108690131B CN 201810558302 A CN201810558302 A CN 201810558302A CN 108690131 B CN108690131 B CN 108690131B
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- kangaroo skin
- kangaroo
- collagen peptide
- skin collagen
- enzymolysis
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- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of biological products, in particular to kangaroo skin collagen peptide, wherein the average molecular weight of the kangaroo skin collagen peptide is below 1KDa, and the kangaroo skin collagen peptide is prepared by the following steps: boiling the kangaroo skin in double-distilled water and boiling water, removing hair and fat, and carrying out enzymolysis on the kangaroo skin by adopting protease to obtain an enzymolysis liquid; and (4) centrifuging the enzymolysis liquid, taking supernatant, freezing and drying to obtain the kangaroo skin collagen peptide. The kangaroo skin collagen peptide can improve the added value of kangaroo production and processing, can reduce environmental pollution and obtain good social and economic benefits. The kangaroo skin collagen peptide has remarkable effects in three aspects of oxidation resistance, in-vitro moisture retention and whitening, and can be used as an antioxidant, a humectant and a whitening agent in cosmetics.
Description
Technical Field
The application relates to the technical field of biological products, in particular to kangaroo skin collagen peptide and application thereof as an antioxidant, a humectant and a whitening agent of cosmetics.
Background
Collagen is the most abundant protein in mammals and accounts for 25-33% of the total protein content in the body. Collagen is a main component of animal connective tissues, is mainly present in tissues such as animal skin, bones and tendons, and plays a role in supporting and protecting. The diversity and complexity of the structural and functional characteristics of collagen protein determine the important position of the collagen protein in many fields and good application prospect.
The collagen peptide is a novel collagen product and is produced by taking collagen or a substance rich in collagen as a raw material. It is the product of collagen protein after hydrolysis reaction under certain external condition. The external conditions are: the collagen is a stable ternary spirochete, which is a product obtained by decomposing and breaking molecular chains under the chemical action or the action of bacteria and enzyme and processing. It has the characteristic amino acid composition of collagen and small molecular weight. Therefore, it is easier to be absorbed by the dermis through the epidermis and is also easier to be digested through the digestive tract, and the application in the fields of daily chemicals and food health care is very good.
Regarding the preparation of collagen peptide, studies on obtaining collagen peptide from the skin, scale and bone of fish or the skin and bone of pig and cow by enzymolysis are now commonly found. In addition, in the prior art, when extracting collagen from raw materials, it is usually necessary to extract collagen by acidolysis or enzymolysis, and then select a suitable protease to enzymolyze collagen into small molecule peptides, so as to finally obtain collagen peptides. Collagen has a large molecular weight, is hardly soluble in water and only soluble in acid, and the dissolution mode of collagen is greatly limited. Therefore, the enzymolysis of collagen needs to be performed under acidic conditions, which greatly influences the selection of enzyme types, and the enzymolysis of collagen has high requirements on pH, and only a few enzymes (such as acidic protease like pepsin) can be selected. The enzymolysis time is long, the effect is poor, the yield is low, and the extraction process is complicated. However, collagen peptide using kangaroo skin as a raw material has not been reported yet. Compared with the collagen fiber bundles of the skin of a common mammal, the collagen fiber bundles of the kangaroo skin have different weaving forms, most of the collagen fiber bundles are parallel to the skin surface and woven in a wave-shaped layered manner, different layers are mutually connected in a staggered manner, the staggered angle between the layers is smaller than 90 degrees, the weaving forms of the collagen fibers of all parts are basically the same, and the elastic fibers of the kangaroo skin are smaller but are distributed more uniformly. Due to the unique fiber structure, the leather care product is not only a good leather product, but also a potential resource of a beauty product. At present, the research on kangaroo skin is still at the level of manufacturing kangaroo skin glue and leather products, and the development of kangaroo skin glue and leather products is not complete. Therefore, the development and application of kangaroo skin become a focus and a hot spot, and huge economic benefits and social benefits can be generated.
Disclosure of Invention
The invention provides kangaroo skin collagen peptide, wherein the average molecular weight of the kangaroo skin collagen peptide is below 1.0 KDa.
The invention also provides a preparation method of the kangaroo skin collagen peptide, which comprises the following steps:
(1) cooking the dehaired and degreased kangaroo skin in boiling water;
(2) carrying out enzymolysis treatment on the unhaired and degreased kangaroo skin by adopting protease to obtain enzymolysis liquid;
(3) and carrying out centrifugal separation on the enzymolysis liquid, and drying the supernatant to obtain the kangaroo skin collagen peptide.
In the step (1) of the preparation method of the kangaroo skin collagen peptide, the kangaroo skin is cooked in boiling water for 20-40 minutes, and the amount of the boiling water is 10-30 times of the volume (ml/g) of the weight of the unhaired and degreased kangaroo skin. The water is preferably double distilled water, because the double distilled water has less impurities. Of course, other clean waters may be used.
In the step (1) of the preparation method of the kangaroo skin collagen peptide, the amount of the boiling water is 15-25 times of the volume (ml/g) of the weight of the kangaroo skin after unhairing and defatting.
In the step (2) of the method for preparing kangaroo skin collagen peptide, the protease may be any one of acid protease, alkaline protease and neutral protease. Preferably, the protease is selected from one or more of papain, pepsin and pancreatin, preferably papain. More preferably, the weight ratio of the papain to the kangaroo skin is 1% -5%, more preferably 2%.
The invention tries to mix two enzymes for use, takes ABTS free radical clearance rate as an index, and obtains the optimal conditions of the mixed enzyme through orthogonal experiments. However, the enzymolysis of the mixed enzyme needs long time and the process is complicated, and the obtained effect is comparable to that of the single-use papain, so only the papain is finally selected. The optimum conditions for the enzymatic hydrolysis vary depending on the kind of enzyme.
In the step (2) of the preparation method of kangaroo skin collagen peptide, when papain is used for enzymolysis, the enzymolysis conditions are as follows: the weight ratio of papain to kangaroo skin is 1-2%, the pH is 5.4-9.4, the temperature is 40-55 ℃, and the enzymolysis time is 2-5 hours. The enzymolysis time cannot be too short, and the enzymolysis is not complete if the time is too short. Too long is not only unnecessary, but also has some negative effect on the scavenging of free radicals.
In the step (3) of the preparation method of kangaroo skin collagen peptide, the centrifugation condition is to centrifuge at 4000-.
In the above-mentioned step (3) of the method for preparing kangaroo skin collagen peptide, the drying includes freeze-drying and spray-drying, preferably freeze-drying.
The invention also discloses application of the kangaroo skin collagen peptide as an antioxidant, a whitening additive and a humectant in cosmetics.
The invention also discloses an antioxidant, a whitening additive and a humectant for cosmetics, wherein the antioxidant, the whitening additive and the humectant comprise the kangaroo skin collagen peptide.
The invention also discloses an antioxidant, a whitening additive and a humectant for cosmetics, wherein the antioxidant, the whitening additive and the humectant comprise the kangaroo skin collagen peptide.
The kangaroo skin contains collagen, and can obtain high-quality collagen peptide after being treated by a proper method of modern enzymology biotechnology. The preparation method of the kangaroo skin collagen peptide disclosed by the application is different from the traditional complex process for treating raw materials, but skips the process of extracting collagen from the raw materials, directly treats the raw materials, obtains high-quality collagen peptide with good effect, and achieves the purpose of simplifying the preparation process. The kangaroo skin collagen peptide prepared by the method can improve the additional value of kangaroo production and processing, can reduce environmental pollution and obtain good social and economic benefits. Experiments show that the kangaroo skin collagen peptide has remarkable effects in three aspects of oxidation resistance, in-vitro moisture retention and whitening. In an experiment that a human HepG2 liver cancer cell is used as a hydrogen peroxide modeling cell, the kangaroo skin collagen peptide has improved cell oxidation resistance and has a good effect of clearing active oxygen in the cell. The kangaroo skin collagen peptide has a good inhibition effect on tyrosinase in vitro, can effectively reduce the activity of the tyrosinase in B16 melanocytes, and has a good whitening effect.
Drawings
FIG. 1 shows the effect of neutral protease on ABTS clearance under different enzymatic conditions, using ABTS free radical clearance as an indicator;
FIG. 2 shows the effect of acidic proteases on ABTS clearance under different enzymatic conditions, using ABTS free radical clearance as an indicator;
FIG. 3 shows the effect of alkaline protease on ABTS clearance under different enzymatic conditions, using ABTS free radical clearance as an indicator;
FIG. 4 shows the effect of papain on ABTS clearance under different enzymatic conditions, using ABTS free radical clearance as an indicator;
FIG. 5 shows the effect of pepsin under different enzymatic conditions on ABTS clearance, using ABTS free radical clearance as an indicator;
FIG. 6 shows the effect of trypsin on ABTS clearance under different enzymatic conditions, using ABTS free radical clearance as an indicator;
FIG. 7 shows the effect on yield and ABTS clearance under optimal enzymatic conditions for different proteases; wherein the abscissa is the type of protease (neutral protease, alkaline protease, acid protease, papain, pepsin, trypsin), and the left and right ordinates are the protein polypeptide yield and ABTS clearance respectively;
FIG. 8 shows a MALDI-TOF MS spectrum of kangaroo skin collagen peptide of the present application;
fig. 9 shows the uv absorption peak of kangaroo skin collagen peptide of the present application;
FIG. 10 shows the moisturizing ability of Kangaroo skin collagen peptides of the present application, in which ABC represents the added concentration of Kangaroo skin polypeptides: peptide A (low) 10 mg/ml; peptide B (middle) 100 mg/ml; c peptide (high) 1 g/ml);
FIGS. 11 to 13 show the inhibition of tyrosinase in vitro by kangaroo skin collagen peptides prepared by the method of the present invention; the kangaroo skin collagen polypeptide prepared by the method of the invention has the advantages that the inhibition on tyrosinase diphenolase and monophenolase is irreversible; FIG. 11 shows the inhibition of tyrosinase diphenolase in vitro by kangaroo skin collagen peptides of the present application, with increasing peptide concentration, decreasing enzyme activity; FIG. 12 shows the inhibition pattern of kangaroo skin collagen peptide on tyrosinase diphenolase in vitro prepared by the method of the present invention, and it can be seen that the inhibition pattern is irreversible inhibition; FIG. 13 shows the inhibition of tyrosinase monophenolase in vitro by kangaroo collagen peptides of the present application, with increasing peptide concentration, time delay of the reaction, and decreasing reaction products;
FIG. 14 shows the effect of kangaroo skin collagen peptide prepared by the method of the present invention on the proliferation rate in mouse B16 melanoma cells; the abscissa is the kangaroo skin collagen peptide treatment groups with different concentrations (the concentrations of kangaroo skin collagen peptide are 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mug/muL respectively), and the ordinate is the cell survival rate;
FIG. 15 shows the effect of kangaroo skin collagen peptide prepared by the method of the present invention on tyrosine activity in mouse B16 melanoma cells; the abscissa is the kangaroo skin collagen peptide treatment groups with different concentrations (the concentrations of kangaroo skin collagen peptide are 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mug/muL respectively), and the ordinate is the relative activity of tyrosine activity in cells;
FIG. 16 shows superoxide anion radical scavenging ability of Kangaroo skin collagen peptide prepared by the method of the present invention; each curve represents the concentration of kangaroo skin collagen peptide (0, 0.5, 1, 1.5 mug/muL);
FIG. 17 shows the effect of kangaroo collagen peptide prepared by the method of the present invention on the proliferation rate of human liver cancer HepG2 cells; the abscissa is the kangaroo skin collagen peptide treatment groups with different concentrations (the concentrations of kangaroo skin collagen peptide are 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mug/muL respectively), and the ordinate is the relative cell proliferation rate;
FIG. 18 shows the effect of hydrogen peroxide on the morphology of human liver cancer HepG2 cells; a, B, C, D, E, F in the figure are A: normal values (no drug added); b: 100. mu. mol of hydrogen peroxide; c: 200 mu mol/L hydrogen peroxide; d: 300 mu mol/L hydrogen peroxide; e: 400 μmol/L hydrogen peroxide; f: 500. mu. mol/L hydrogen peroxide;
FIG. 19 shows the effect of Kangaroo skin collagen peptides prepared by the method of the present invention on hydrogen peroxide damage model cell morphology; a, B, C, D, E, F in the figure are respectively: a: model group (600. mu. mol/L hydrogen peroxide); b: a normal group; c: 600 μmol/L hydrogen peroxide +0.1 μ g/μ L peptide; d: 600 μmol/L hydrogen peroxide +0.2 μ g/μ L peptide; e: 600 μmol/L hydrogen peroxide +0.3 μ g/μ L peptide; f600. mu. mol/L hydrogen peroxide + 0.4. mu.g/. mu.L peptide);
FIGS. 20 to 22 show the effect of kangaroo collagen peptide prepared by the method of the present invention on hydrogen peroxide-damaged Reactive Oxygen Species (ROS) content in HepG2 cells of human liver cancer; the test included a control group (FIG. 20) administered at a dose of 0. mu.g/. mu.L, 600. mu. mol/L H2O2+ 0.50. mu.g/. mu.L kangaroo skin polypeptide FIG. 21), 600. mu. mol/L H2O2(FIG. 22); the G-MEAN values were 105.6 (FIG. 20), 118.94 (FIG. 21), and 221.3 (FIG. 22), respectively.
Detailed Description
Condition study of neutral protease enzymolysis kangaroo skin
After the kangaroo skin is shaved, macroscopic white fat is cut off by scissors, the kangaroo skin is cleaned and drained, then 10g of kangaroo skin is taken and added with double distilled water (200mL) with 20 times of volume, and the rice cooker is stewed for 20 min. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. 0.1g of neutral protease (the enzyme activity of Anhui ferbamei production is 100u/mg) is added into each part, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 2 hours. The pH values of the enzymolysis are 4.4, 5.4, 6.4, 7.4 and 8.4 respectively. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 1, it is demonstrated that the activity of the enzyme-hydrolyzed kangaroo skin is highest and the enzyme-hydrolyzed effect is best when the pH value is 7.4 for neutral protease.
After the kangaroo skin is shaved, macroscopic white fat is cut off by scissors, the kangaroo skin is cleaned and drained, then 10g of kangaroo skin is taken and added with double distilled water (150mL) with 15 times of volume, and the rice cooker is stewed for 40 min. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. Adding 0.1g neutral protease into each part, and performing enzymolysis at pH of 7.4 for 2 h. The enzymolysis temperature is 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, and 55 deg.C. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 1, it is demonstrated that the activity of zymolysis of kangaroo skin is highest and the zymolysis effect is best when the temperature is 50 ℃ for neutral protease.
After the kangaroo skin is shaved, macroscopic white fat is cut off by scissors, the kangaroo skin is cleaned and drained, then 10g of kangaroo skin is taken and added with double distilled water (250mL) with the volume being 25 times of the volume of the kangaroo skin, and the kangaroo skin is stewed for 30min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. After each portion is adjusted to the optimum pH of 7.4, 0.1g of protease is added, and enzymolysis is carried out at 50 ℃. The enzymolysis time is 2h, 3h, 4h, 5h and 6 h. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in figure 1, the activity of the kangaroo skin is highest and the enzymolysis effect is best when the time is 5 hours for the neutral protease.
Shaving Kangaroo skin, cutting off macroscopic white fat with scissors, cleaning, draining, adding 10g Kangaroo skin into 10, 15, 20, 25, 30 times volume of double distilled water, and cooking with electric cooker for 30 min. Cooling to room temperature, and mincing. Adjusting pH of each part to 7.4, adding 0.1g protease into each part, performing enzymolysis at 50 deg.C for 5 hr, and removing ABTS free radical with hydrolysate as index. As shown in figure 1, the enzyme digestion activity of kangaroo skin is highest and the enzyme digestion effect is best when the feed-liquid ratio is 1:10 for neutral protease.
Acid proteaseCondition study of enzymolysis kangaroo skin
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. 0.2g of acid protease (50 u/mg of enzyme activity produced by Anhui Hefei Bomei) is added into each part, the enzymolysis temperature is 45 ℃, and the enzymolysis time is 2 hours. The pH values of the enzymolysis are 2.4, 3.4, 4.4, 5.4 and 6.4 respectively. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 2, it is demonstrated that the acidic protease has the highest activity and the best enzymolysis effect when the pH value is 3.4-4.4.
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. Adding 0.2g of acid protease into each part, wherein the enzymolysis pH is 3.4, and the enzymolysis time is 2 h. The enzymolysis temperature is 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, and 55 deg.C. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 2, it is demonstrated that the acidic protease has the highest activity and the best enzymolysis effect when the temperature is 35 ℃.
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. After adjusting the optimum pH to 3.4, 0.2g protease was added to each portion, and the mixture was subjected to enzymatic hydrolysis at 35 ℃. The enzymolysis time is 2h, 3h, 4h, 5h and 6 h. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 2, it is demonstrated that the activity of the kangaroo skin is the highest and the enzymolysis effect is the best when the enzymolysis time is 6 hours for the acid protease.
Adding 10g kangaroo skin (removing fat after shaving and draining) into 10, 15, 20, 25 and 30 times volume of double distilled water respectively, and cooking with electric cooker for 40 min. . Cooling to room temperature, and mincing. After each part is adjusted to the optimum pH value of 3.4, 0.2g protease (accounting for 2% of the weight of kangaroo skin) is added, and enzymolysis is carried out for 6h at 35 ℃ by taking ABTS free radicals removed by hydrolysate as indexes. As shown in FIG. 2, it is demonstrated that the acidic protease has the highest activity and the best enzymolysis effect when the feed-liquid ratio is 1: 20.
Condition research of alkaline protease enzymolysis kangaroo skin
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. 0.1g of alkaline protease (the enzyme activity of Anhui ferbamei production is 200u/mg) is added into each part, the enzymolysis temperature is 40 ℃, and the enzymolysis time is 2 hours. The pH values of the enzymolysis are 8.4, 9.4, 10.4, 11.4 and 12.4 respectively. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 3, it is demonstrated that the alkaline protease has the highest activity and the best enzymolysis effect on kangaroo skin at pH 11.4.
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. Adding 0.1g of alkaline protease into each part, and performing enzymolysis at pH of 11.4 for 2 h. The enzymolysis temperature is 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, and 55 deg.C. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 3, it is demonstrated that the activity of the enzyme-hydrolyzed kangaroo skin is the highest and the enzyme-hydrolyzed effect is the best at 40 ℃ for the alkaline protease.
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. Adding 0.1g of alkaline protease into each portion, performing enzymolysis at 40 deg.C with pH of 11.4. The enzymolysis time is 2h, 3h, 4h, 5h and 6 h. ABTS free radical scavenging by hydrolysate is taken as an index. The results are shown in FIG. 3. The results show that the enzymolysis time is 4h, the activity of the kangaroo skin is highest, and the enzymolysis effect is best for the alkaline protease.
Adding 10g kangaroo skin (removing fat after shaving and draining) into 10, 15, 20, 25 and 30 times volume of double distilled water, and cooking with electric cooker for 20 min. Cooling to room temperature, and mincing. Adding 0.1g of alkaline protease into each part, performing enzymolysis at 40 deg.C for 4h with pH of 11.4, and removing ABTS free radicals with hydrolysate as index. As shown in FIG. 3, it is demonstrated that the alkaline protease has the highest activity and the best enzymolysis effect when the feed-liquid ratio is 1: 15.
Condition study of papain enzymolysis kangaroo skin
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. Adding 0.1g papain (3500 u/mg enzyme activity in production) into each part, and performing enzymolysis at 55 deg.C for 2 hr. The pH values of the enzymolysis are 5.4, 6.4, 7.4, 8.4 and 9.4 respectively. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 4, it is demonstrated that, in the case of papain, the activity of the enzyme-hydrolyzed kangaroo skin is the highest at pH7.4, and the enzyme-hydrolyzed effect is the best.
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. Adding 0.3g of papain into each part, and carrying out enzymolysis for 2 hours at pH 6.4. The enzymolysis temperature is 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, and 60 deg.C. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 4, it is demonstrated that the papain has the highest enzymolysis activity and the best enzymolysis effect on kangaroo skin at 50 ℃.
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. Adding papain 0.1g per part, performing enzymolysis at 50 deg.C and pH of 6.4. The enzymolysis time is 2h, 3h, 4h, 5h and 6 h. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 4, the papain was digested for 4 hours, and the digestion effect of kangaroo skin was the best.
Adding 10g kangaroo skin (removing fat after shaving and draining) into 10, 15, 20, 25 and 30 times volume of double distilled water respectively, and cooking with electric cooker for 40 min. Cooling to room temperature, and mincing. Adding 0.1g of papain into each part, performing enzymolysis at 50 ℃ for 4h with pH of 6.4, and removing ABTS free radicals with hydrolysate as index. As shown in FIG. 4, it is demonstrated that the papain has the highest enzymolysis activity and the best enzymolysis effect when the feed-liquid ratio is 1: 20.
Condition study of pepsin enzymolysis kangaroo skin
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. 0.1g of pepsin (3500 u/mg of enzyme activity in the production) is added into each part, the enzymolysis temperature is 35 ℃, and the enzymolysis time is 2 hours. The pH values of the enzymolysis are 2.4, 3.4, 4.4, 5.4 and 6.4 respectively. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 5, it is demonstrated that, in the case of pepsin, the activity of the kangaroo skin is the highest at pH 2.4, and the enzymolysis effect is the best.
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. Adding 0.4g of pepsin into each part, and carrying out enzymolysis for 2 hours, wherein the pH value is 2.4. The enzymolysis temperature is 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, and 50 deg.C. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 5, it is demonstrated that, in the case of pepsin, the activity of the kangaroo skin is the highest and the enzymolysis effect is the best at 35 ℃.
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. Adding 0.1g pepsin into each part, performing enzymolysis at 35 deg.C and pH of 2.4. The enzymolysis time is 2h, 3h, 4h, 5h and 6 h. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 5, it is demonstrated that, in the case of pepsin, the activity of the kangaroo skin is the highest and the enzymolysis effect is the best when the time is 4 hours.
Adding 10g kangaroo skin (removing fat after shaving and draining) into 10, 15, 20, 25 and 30 times volume of double distilled water, and cooking with electric cooker for 20 min. Cooling to room temperature, and mincing. Adding 0.1g of pepsin into each part, carrying out enzymolysis at the pH of 2.4 for 4 hours at 35 ℃, and taking ABTS free radicals removed by hydrolysate as an index. As shown in FIG. 5, it is demonstrated that, for pepsin, the activity of the kangaroo skin is highest and the enzymolysis effect is best when the feed-liquid ratio is 1: 20.
Condition study of trypsin enzymolysis kangaroo skin
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. 0.1g of trypsin (the enzyme activity of the industrial production is 250u/mg) is added into each part, the enzymolysis temperature is 35 ℃, and the enzymolysis time is 2 hours. The pH values of the enzymolysis are 5.4, 6.4, 7.4, 8.4 and 9.4 respectively. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 6, it is demonstrated that, in the case of trypsin, the activity of the enzyme-hydrolyzed kangaroo skin is the highest at pH6.4, and the enzyme-hydrolyzed effect is the best.
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. 0.1g of trypsin is added into each portion, the pH value of enzymolysis is 6.4, and the enzymolysis time is 2 hours. The enzymolysis temperature is 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, and 50 deg.C. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 6, it is demonstrated that the activity of the enzyme-hydrolyzed kangaroo skin is highest and the enzyme-hydrolyzed effect is best when the temperature is 35 ℃ for trypsin.
10g Kangaroo skin (defatting after shaving and draining) is added with double distilled water (200mL) with the volume 20 times of that of the Kangaroo skin, and the Kangaroo skin is cooked for 20min by an electric cooker. Cooling to room temperature, and mincing. The mixture was divided into 5 portions in equal amounts. Adding 0.1g of trypsin into each portion, and performing enzymolysis at 35 ℃ and pH of 6.4. The enzymolysis time is 2h, 3h, 4h, 5h and 6 h. ABTS free radical scavenging by hydrolysate is taken as an index. As shown in FIG. 6, it is demonstrated that the activity of the enzyme-hydrolyzed kangaroo skin is the highest and the enzyme-hydrolyzed effect is the best when the time is 4 hours for trypsin.
Adding 10g Kangaroo skin (defatted and drained Kangaroo skin after shaving) into 10, 15, 20, 25, 30 times volume of double distilled water, and cooking with electric cooker for 20 min. Cooling to room temperature, and mincing. Adding 0.5g of trypsin into each part, carrying out enzymolysis at 35 ℃ for 4h with the pH of 6.4, and taking ABTS free radicals removed by hydrolysate as an index. As shown in FIG. 6, it is demonstrated that the activity of the enzyme-hydrolyzed kangaroo skin is highest and the enzyme-hydrolyzed effect is best when the feed-liquid ratio is 1:20 for trypsin.
Optimization of extraction conditions of kangaroo skin collagen peptide
Extracting kangaroo skin protein polypeptide under the optimum conditions of neutrality, acidity, alkalinity, pawpaw and trypsin. The polypeptide yield and ABTS clearance were plotted on the ordinate and the enzyme type on the abscissa. The results are shown in FIG. 7, where the best enzymatic ability of papain was obtained.
Table 1 shows an orthogonal experiment of papain enzymatic hydrolysis of kangaroo skin. The ABTS clearance is used as a measurement index, the optimal extraction conditions are that the feed-liquid ratio is 1:20, the pH is 6.4, 2 percent (140000u) of enzyme is added, and hydrolysis is carried out for 3 hours +/-10 minutes at the temperature of 55 +/-0.5 ℃.
TABLE 1
Average molecular weight determination of kangaroo skin collagen peptide
0.1% TFA (trifluoroacetic acid) is prepared by taking 50% CAN (acetonitrile) as a solvent, and 1ml of ultrasonic re-dissolved peptide powder is taken. Sucking 1 μ L of redissolved peptide solution and 1 μ L of α -HCCA (α -cyano-4-hydroxycinnamic acid) matrix solution, mixing them uniformly, spotting them on a mass spectrometer sample target, volatilizing the sample matrix, and crystallizing, and using matrix assisted laser analysis ionization time of flight mass spectrometer (MALDI-TOF MS) to make on-machine analysis.
Metal element analysis of kangaroo skin collagen peptide
0.2g of kangaroo skin collagen peptide powder is weighed, 8mL of 65% -68% nitric acid is added, and standing is carried out for 30 min. After microwave digestion for 3 hours, standing and cooling. And (5) using double distilled water to fix the volume to 250mL, so that the final concentration of the nitric acid is 2-3%.
ICP-MS (inductively coupled plasma mass spectrometry) was used for metal element analysis, and table 2 shows the content of metal elements in the kangaroo skin collagen peptide of the present application.
TABLE 2
Amino acid analysis of Kangaroo skin collagen peptide
10g of kangaroo skin collagen peptide powder is weighed, and the total amount of amino acids and the content of 16 amino acids are analyzed by adopting the method of GB 5009.124-2016. Table 3 shows the total amount of kangaroo skin collagen peptide amino acids and the content of 16 amino acids of the present application.
TABLE 3
Example 1
10g of drained kangaroo skin was shaved. Cutting into small pieces of about 1cm by 1cm, adding 20 times volume (200ml) of double distilled water, and cooking with electric cooker for 20 min. Cooling to room temperature, and mincing. Adjusting pH to 6.4, adding 2% papain, and hydrolyzing at 55 + -0.5 deg.C for 3 hr + -10 min. The enzyme was inactivated by boiling for 10 minutes. Centrifugation was carried out at 5000rpm for 30. + -.5 minutes at room temperature. And taking the supernatant, and freeze-drying to obtain kangaroo skin collagen peptide powder.
FIG. 8 shows MALDI-TOF MS spectrum of Kangaroo skin collagen peptide obtained in example 1. The molecular weight of the kangaroo skin collagen peptide obtained in example 1 is below 1 KDa. The smaller molecular weight indicates that the enzymatic hydrolysis was sufficient when papain was used for treatment.
Example 2
10g of drained kangaroo skin was shaved. Cutting into small pieces of about 1cm by 1cm, adding 20 times volume (200ml) of double distilled water, and cooking with electric cooker for 20 min. Cooling to room temperature, and mincing. Adjusting pH to 6.4, adding 2% papain, and hydrolyzing at 55 + -0.5 deg.C for 3 hr + -10 min. The enzyme was inactivated by boiling for 10 minutes. Centrifugation was carried out at 5000rpm for 30. + -.5 minutes at room temperature. And taking the supernatant, and freeze-drying to obtain kangaroo skin collagen peptide powder.
FIG. 9 shows that the aqueous solution of Kangaroo skin collagen peptide obtained in example 2 has a maximum absorption peak around 220nm after ultraviolet full-wavelength scanning. This is the characteristic absorption peak of collagen.
Example 3
The in vitro moisturizing effect of kangaroo skin collagen peptide
According to the difference of the moisture retention performance of the humectant, the acting force of the humectant with different concentrations on water molecules is different, and the capacities of absorbing water and retaining water are also different. The sample containing certain moisture is put in a dryer with constant temperature and humidity for drying, the reduction of the sample mass is weighed at regular time, and the moisture retention of different samples can be compared through comparative analysis. To better simulate the actual condition of the skin, the slides were taped with 3M, and each test item (kangaroo skin peptide (low concentration) 10 mg/ml; peptide (medium concentration) 100 mg/ml; peptide (high concentration) 1g/ml) was applied to it, placed in a desiccator containing a saturated ammonium sulphate solution (85% relative humidity), weighed every 2 hours, and the water loss rate for that period was calculated, and the results are shown in FIG. 12. The water loss rate calculation formula is:
water loss (%) - (m1-m2)/m 1X 100%
Where m1 is the initial moisture mass of the sample and m2 is the moisture mass of the sample after being left for 2 hours.
The results show that of the three kangaroo skin polypeptides of the present application, the peptide (low) 10mg/ml has the best moisturizing effect.
Example 4
Whitening effect of Kangaroo skin collagen peptide
1. Half inhibition rate of kangaroo skin collagen peptide on tyrosinase in vitro
The action of kangaroo skin collagen peptide on tyrosinase diphenolase is measured, and the half inhibition rate of the kangaroo skin collagen peptide is researched. The assay used a 3mL system. The effectors were dissolved in water to make solutions containing varying concentrations of the effectors. 0.5mmol/L L-DOPA is taken as a substrate, 0.1mL of effector with different concentrations is firstly added into a cuvette in a 3mL activity measuring system of 0.05mol/L phosphate buffer solution (pH 6.8), 2.8mL of substrate solution which is preserved in a constant temperature water bath at 30 ℃ is then added, 0.1mL of mushroom tyrosinase solution is then added and mixed evenly, the linear increase of the optical density value with time of 475nm at the constant temperature of 30 ℃ is measured, and the enzyme activity is obtained from the slope of the linear. The relative residual activity of the enzyme is plotted against the concentration of the effector to obtain a concentration-effect curve of the effector, and the IC50 value of the effector is obtained from the corresponding concentration of the effector when the relative residual activity of the enzyme is 50%. FIG. 10 shows the IC50 of kangaroo skin collagen peptide of the present application against tyrosinase at 55 mg/ml.
2. Inhibition type of kangaroo skin collagen peptide on tyrosinase in vitro
IC50 values obtained according to the above procedure. The amount of added enzyme protein was varied and the effect of different concentrations of effector on the oxidative activity of mushroom tyrosinase-catalyzed L-DOPA (levodopa) was determined. And (4) mapping the residual enzyme activity of the enzyme after the action of the effector on the added enzyme amount, thereby judging the inhibition action mechanism of the effector on the mushroom tyrosinase diphenolase. Fig. 13 shows that the inhibitory mechanism of kangaroo skin collagen peptide of the present application against tyrosinase diphenolase is irreversible inhibition.
3. Analysis of tyrosinase monophenolase activity by Kangaroo skin collagen polypeptide
The monophenolase activity of tyrosinase is determined by taking L-Tyrosine as a substrate and determining the influence of effectors on the kinetic curve of the mushroom tyrosinase monophenolase catalytic reaction. A3 mL system is adopted, 2mmol/L (final concentration) Tyr (tyrosinase monophenolase substrate) is taken as a substrate, in a living detection system of 3mL 0.05mol/L phosphate buffer solution (pH 6.8) (final concentration), 0.1mL of kangaroo skin collagen polypeptide containing different concentrations is added into a cuvette, 2.8mL of 0.05mol/L phosphate buffer solution is added, a substrate solution preserved in a constant-temperature water bath at 30 ℃ in advance is added, then 100 mu L of mushroom tyrosinase is added, the mixture is immediately mixed, and a linear growth of an optical density value with time of 475nm wavelength is detected under the constant temperature condition of 30 ℃. The results are shown in FIG. 13.
4. Effect of kangaroo skin collagen peptide on proliferation of mouse B16 melanoma cells
Figure 11 shows the effect of kangaroo skin collagen peptides of the present application on mouse B16 melanoma intracellular proliferation. Using RPMI-1640 medium (containing 10% newborn calf serum, penicillin 100U/mL, streptomycin 100. mu.g/mL) in CO2Incubator 37 ℃ CO2Cells were cultured under saturated humidity conditions of 5%. When the cells grow to a near-confluent state, the cells are digested by 0.25 percent trypsin and collected, the concentration of the cells is adjusted, a single cell suspension of mouse B16 melanoma cells is added into a 96-well cell culture plate, 200 mu L of each well is kept overnight, and after the cells adhere to the wall, the culture solution is discarded. Simultaneously adding culture solution containing kangaroo skin polypeptide with different concentrations, continuously culturing for 24h, adding 20 μ L of 0.5mg/mL MTT (4, 5-dimethylthiazole-2) into each well (keeping out of the sun), adding into the culture solution, and culturingCO2Incubator 37 ℃ CO2After culturing the cells under the condition of 5% of saturated humidity for 4h, removing the supernatant, adding 180 mu L of DMSO into each well, shaking the mixture at room temperature in the dark for about 10min to completely dissolve formazan crystals, and immediately measuring the light absorption value at 570nm by an enzyme labeling instrument. For each concentration, 5 replicate wells were set and averaged. The same passage cells were taken for each experiment.
The abscissa represents the kangaroo skin collagen peptide treatment groups at different concentrations (the concentrations of kangaroo skin collagen peptide are 0, 0.1, 0.2, 0.3, 0.4 and 0.5. mu.g/. mu.L, respectively), and the ordinate represents the relative cell proliferation rate. FIG. 14 illustrates that the kangaroo skin collagen peptide has no toxic effect on the melanoma cells of the mouse B16 in the range of 0-0.5. mu.g/. mu.L.
5. Effect of kangaroo skin collagen peptide on tyrosine activity in mouse B16 melanoma cells
The cell culture method was the same as above. After the cells were grown to near confluent state, they were collected by 0.25% trypsin digestion and adjusted for cell concentration, 2X 104Inoculating in a 6-well plate, standing overnight, changing the liquid after the wall is adhered, and respectively adding 2mL of culture medium containing 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mu g/mu L of kangaroo skin collagen peptide. The culture was continued for 24 hours and then the culture solution was discarded. After washing 2 times with PBS buffer pH7.4, 900. mu.L of PBS buffer containing 1% Triton X-100 by volume fraction was added to each well, 100. mu.L of 1.0mg/mL L-DOPA was added thereto, and sonication was carried out for 2 minutes, followed by incubation at 30 ℃ for 30 minutes, and the absorbance at 475nm was measured. Each concentration treatment was set to 3 replicates and averaged. The same passage cells were taken for each experiment.
Figure 12 shows the effect of kangaroo skin collagen peptides of the present application on tyrosine activity in mouse B16 melanoma cells. Under the action of kangaroo skin collagen peptide with the concentration range of 0-0.5 mug/muL, the tyrosine activity in the melanoma cells of the mouse B16 shows a trend of decreasing along with the increase of the concentration of the kangaroo skin collagen peptide.
Example 5
Antioxidant capacity of kangaroo skin collagen peptide
1. Scavenging capacity of kangaroo skin collagen peptide on superoxide anion free radicals
The scavenging capacity of kangaroo skin collagen peptides for superoxide anion free radicals is measured by the pyrogallol autoxidation rate. Mixing Tris-HCl buffer solution (50mmol/L, pH 8.2, 4.5mL) with pure water (4.2mL), incubating for 20min at 25 ℃, quickly adding 0.3mL pyrogallol solution (3mmol/L, prepared by 10mmol/L hydrochloric acid and preheated at 25 ℃), uniformly mixing, recording the absorbance change of the reaction solution at 320nm within 5min, drawing a time (t) -absorbance (A) curve, and calculating the absorbance change in the reaction unit time within a linear range to obtain the autoxidation rate of the pyrogallol.
And (3) measuring the self-oxidation rate of the pyrogallol under the action of the sample to be measured: mixing Tris-HCl buffer solution (50mmol/L, pH 8.2, 4.5mL) with pure water (3.3mL), incubating for 20min at 25 ℃, quickly adding 0.9mL sample and 0.3mL pyrogallol solution (3mmol/L, prepared by 10mmol/L hydrochloric acid and preheated at 25 ℃), uniformly mixing, recording the absorbance change of the reaction solution at 320nm within 5min, drawing a time (t) -absorbance (A) curve, and calculating the absorbance change in the reaction unit time within a linear range to obtain the autoxidation rate of the pyrogallol under the action of the test substance.
Superoxide anion radical scavenging rate (%) - (1-V)1/V0)*100％
V0: pyrogallol autoxidation rate
V1: is the autoxidation rate of pyrogallol under a sample to be measured
From FIG. 13, it is understood that the autoxidation rate of pyrogallol is decreased and the superoxide anion radical scavenging rate is increased with the increase of the concentration of the kangaroo collagen peptide. The result shows that the kangaroo skin collagen peptide has good free radical scavenging capacity.
2. Scavenging capacity of kangaroo skin collagen peptide on hydroxyl free radical
The hydroxyl radical scavenging capacity of kangaroo skin collagen peptide is measured by a salicylic acid competition hydroxyl radical capturing method. 1mL of kangaroo skin collagen peptide (0.25, 0.5, 0.75, 1, 1.5. mu.g/. mu.L) with different concentrations and 1mL of ferrous sulfate (FeSO)49mmol/L) and 1mL hydrogen peroxide (10mmol/L), incubating at 37 deg.C for 10min, adding 1mL salicylic acid (9mmol/L), mixing, incubating at 37 deg.CIncubating for 10min, measuring the absorbance of the reaction solution at 510nm, and using pure water as blank control. The OH of the sample to be measured is calculated according to the following formula-Free radical scavenging rate. The results are shown in Table 4.
Hydroxyl radical clearance (%) - (1-a experimental absorbance/a blank absorbance) × 100%
TABLE 4
| Concentration of | 0.25μg/μL | 0.5μg/μL | 0.75μg/μL | 1μg/μL | 1.5μg/μL |
| Clearance rate | 33％ | 44.75％ | 58％ | 70％ | 95％ |
3. DPPH (dipeptidyl peptidase) removing capability of kangaroo skin collagen peptide
DPPH method according to the established method of Brand-Williams et al, 0.1ml of kangaroo skin collagen peptide (0, 2.5, 5, 7.5, 10. mu.g/. mu.L) was taken at various concentrations and 3ml of a 0.004% DPPH methanol solution was added. After mixing uniformly, standing for 30min, and measuring the light absorption value at 517 nm. Methanol was used as a blank. The results are shown in Table 5.
Inhibition ratio (%) [ (A)0－A1)/A0]*100％
Wherein A is0(addition of 0.1mL of methanol and 3mL of DPPH methanol solution) as absorbance value before addition of sample, A1The absorbance after sample addition is used.
TABLE 5
| Concentration of | 0μg/μL | 2.5μg/μL | 5μg/μL | 7.5μg/μL | 10μg/ |
| Clearance rate | |||||
| 0 | 13％ | 23％ | 28％ | 34％ |
4. ABTS free radical scavenging capacity of kangaroo skin collagen peptide
Early preparation: adding distilled water 25mL into ABTS 96mg to prepare solution A; get K2S2O3784mg of the above extract was added with 10mL of distilled water to prepare solution B. Mixing 5ml of the solution A with 88 mu of solution L B uniformly, standing for 12-16 hours, and preparing ABTS working solution.
The ABTS working solution is diluted by PBS solution, and the light absorption value at 734nm at normal temperature is required to be 0.7 +/-0.02. Mixing 0.2ml of LaBTS working solution with 10uL of kangaroo skin collagen peptide (0, 2.5, 5, 7.5 and 10 mug/mu L) with different concentrations, standing for 6min at normal temperature in a dark place, and measuring the absorbance at the wavelength of 734 nm. The results are shown in Table 6.
ABTS free radical clearance (100%) (A0-Ai/A0) × 100%
Wherein A is0Adding ABTS absorbance for no sample; ai is the absorbance of the added sample and ABTS
TABLE 6
| Concentration of | 0μg/μL | 2.5μg/μL | 5μg/μL | 7.5μg/μL | 10μg/ |
| Clearance rate | |||||
| 0％ | 15％ | 32％ | 51％ | 60％ |
Example 6
Application of kangaroo skin collagen polypeptide in antioxidant model
1. The influence of kangaroo skin collagen peptide on the proliferation rate of human liver cancer HepG2 cells.
FIG. 17 shows the effect of kangaroo skin collagen peptide of the present application on the proliferation rate of human liver cancer HepG2 cells. DMEM medium (containing 10% newborn calf serum, penicillin 100U/mL, streptomycin 100. mu.g/mL) in CO2Incubator 37 ℃ CO2Cells were cultured under saturated humidity conditions of 5%. When the cells grow to a near-fusion state, digesting and collecting the cells by 0.25% trypsin, adjusting the cell concentration, adding 200 mu L of human liver cancer HepG2 cell single-cell suspension into a 96-well cell culture plate, standing overnight, and after the cells adhere to the wall, removing the culture solution. Simultaneously adding culture solution containing kangaroo skin polypeptide with different concentrations, continuously culturing for 24h, adding 20 μ L of 0.5mg/mL MTT (protected from light) into each well, and adding into CO2Incubator 37 ℃ CO2After culturing the cells under the condition of 5% of saturated humidity for 4h, removing the supernatant, adding 180 mu L of DMSO into each well, shaking the mixture at room temperature in the dark for about 10min to completely dissolve formazan crystals, and immediately measuring the light absorption value at 570nm by an enzyme labeling instrument. For each concentration, 5 replicate wells were set and averaged. The same passage cells were taken for each experiment.
The abscissa represents the kangaroo skin collagen peptide treatment groups at different concentrations (the concentrations of kangaroo skin collagen peptide are 0, 0.1, 0.2, 0.3, 0.4 and 0.5. mu.g/. mu.L, respectively), and the ordinate represents the relative cell proliferation rate. FIG. 17 shows that the kangaroo skin collagen peptide has no toxic effect on human liver cancer HepG2 cells in the range of 0-0.5. mu.g/. mu.L, and has no influence on the proliferation of human liver cancer HepG2 cells in the concentration range.
2. Establishment of hydrogen peroxide damaged human liver cancer HepG2 cell model
Taking HepG2 cells in a logarithmic growth phase, inoculating the cells in a 6-well plate, culturing for 12h at 37 ℃ in 5% CO2(v/v), discarding old culture solution, adding hydrogen peroxide with different concentrations (0-500 mu M) to treat the HepG2 cells for 24h, treating with Giemsa staining solution, placing the 6-well plate on an inverted phase-contrast microscope, observing the shapes of different groups of cells, and taking pictures. The results are shown in FIG. 18, where control HepG2 cells were full, smooth-edged, well defined, and had fusiform or irregular polygons. In the hydrogen peroxide treated group, the number of cells was reduced, the distribution was loose, and a small amount of cells were rounded.
3. Influence of kangaroo skin collagen peptide on hydrogen peroxide damage model cell morphology
Taking HepG2 cells in logarithmic growth phase, inoculating the HepG2 cells in a 6-well plate, and inoculating the HepG2 cells in the logarithmic growth phase in a 5% CO solution at 37 DEG C2(v/v) culturing for 24h, discarding the old culture solution, adding drugs with different concentrations to treat HepG2 cells for 24h, and discarding the culture solution. Washing with PBS for 2 times, fixing with methanol for 15min, washing with PBS for 2 times, adding 500 μ L of Hochest staining solution into each well, incubating in dark for 10min, discarding the staining solution, observing the form of cell nucleus under a fluorescence inverted microscope, and taking a picture. The results are shown in FIG. 19, where the normal group of HepG2 cells were full, smooth-edged, well defined, and had fusiform or irregular polygons. In the hydrogen peroxide treatment group, the number of cells was reduced, the distribution was loose, and a large number of cells were necrotic. The cells treated by the hydrogen peroxide and the kangaroo skin polypeptide have gradually increased cell number and gradually reduced necrosis number along with the increase of the polypeptide concentration.
4. Effect of kangaroo skin collagen peptide on hydrogen peroxide damage of Reactive Oxygen Species (ROS) content in human liver cancer HepG2 cells
HepG2 cells were seeded in 6-well plates in logarithmic growth phase at 37 ℃ with 5% CO2(v/v) culturing for 24h, discarding the old culture solution, adding drugs with different concentrations to treat HepG2 cells for 24h, and discarding the culture solution. Cells were collected by trypsinization in 1.5mL Ep tubes. The remaining cells were washed once more with 1mL PBS and also collected in the same Ep tube, centrifuged at 2500rpm for 5 minutes, the supernatant removed and then resuspended in 1mL PBS, and the supernatant again centrifuged. After 1mL of PBS had resuspended the cells, 1. mu.L of 10mmol/L probe DCFH-DA (dichlorofluorescein diacetate) was added and incubated at 37 ℃ for 30 minutes. The supernatant was then centrifuged again and the cells were resuspended in 1mL PBS. The cells were analyzed for ROS on a flow cytometer.
FIG. 20 shows the effect of kangaroo skin collagen peptides of the present application on hydrogen peroxide-damaged content of HepG2 reactive oxygen species in human hepatoma cells. From left to right, the control group is 0 mu g/mu L, 600 mu mol/LH2O2And 0.50. mu.g/. mu.L Kangaroo skin collagen polypeptide, 600. mu. mol/LH 202. The G-MEAN values are 105.6, 118.94, 221.3 from left to right, respectively.
A larger G-MEAN value indicates a higher ROS activity. In the presence of hydrogen peroxide and kangaroo collagen peptide, the activity of ROS was higher than that of the control group, but inferior to that of the treatment group in which only hydrogen peroxide was present. The result shows that the kangaroo skin collagen peptide has the capability of clearing active oxygen clusters and can reduce the oxidative damage of cells.
Therefore, the kangaroo skin collagen peptide has good antioxidant effect.
In summary, the above description is only a preferred embodiment of the present application, and is not intended to limit the scope of the present application. Therefore, any modification, equivalent replacement, improvement or the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (7)
1. The kangaroo skin collagen peptide is characterized in that the average molecular weight of the kangaroo skin collagen peptide is below 1 KDa; the kangaroo skin collagen peptide is prepared by the following steps:
(1) cooking the defatted and dehaired kangaroo skin in double-distilled water boiling water; wherein the cooking time in boiling water is 20-40 minutes, and the consumption of the boiling water is 10-30 times of the volume of the degreased and unhaired kangaroo skin in terms of ml/g;
(2) carrying out enzymolysis treatment on the unhaired and degreased kangaroo skin by adopting protease to obtain enzymolysis liquid;
(3) centrifuging the enzymolysis liquid, and freeze-drying or spray-drying the supernatant to obtain kangaroo skin collagen peptide;
in the step (2), the protease is papain, and the enzymolysis conditions are as follows: the weight ratio of papain to kangaroo skin is 1-2%, the pH is 5.4-9.4, the temperature is 40-55 ℃, and the enzymolysis time is 2-5 hours.
2. An antioxidant for cosmetics, comprising the kangaroo skin collagen peptide according to claim 1.
3. A whitening additive for cosmetics, comprising the kangaroo skin collagen peptide according to claim 1.
4. A moisturizing agent for cosmetics, characterized in that the moisturizing agent comprises the kangaroo skin collagen peptide according to claim 1.
5. Use of the kangaroo skin collagen peptide according to claim 1 as an antioxidant in cosmetics.
6. Use of the kangaroo skin collagen peptide according to claim 1 as a whitening additive in cosmetics.
7. Use of the kangaroo skin collagen peptide according to claim 1 as a moisturizer in cosmetics.
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| CN102559826A (en) * | 2012-02-15 | 2012-07-11 | 胡如桂 | Method for preparing collagen oligopeptide |
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| Factors and mechanisms determining the formation of fibrillar collagen structures in adsorbed phases;Elzbieta Gurdak,et al.;《Colloids and Surfaces B: Biointerfaces》;20060930;第52卷(第1期);第1-2页 * |
| 袋鼠皮加工技术;涂颖科等;《中国皮革》;20051231;第34卷(第23期);第76-88页 * |
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