CN108690131A - A kind of kangaroo skin collagen peptide and application thereof - Google Patents
A kind of kangaroo skin collagen peptide and application thereof Download PDFInfo
- Publication number
- CN108690131A CN108690131A CN201810558302.9A CN201810558302A CN108690131A CN 108690131 A CN108690131 A CN 108690131A CN 201810558302 A CN201810558302 A CN 201810558302A CN 108690131 A CN108690131 A CN 108690131A
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- China
- Prior art keywords
- kangaroo
- collagen peptide
- kangaroo skin
- skin collagen
- enzymolysis
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Classifications
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C—CHEMISTRY; METALLURGY
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- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Cosmetics (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to biological product technical fields, and more particularly to a kind of kangaroo skin collagen peptide, the average molecular weight of the kangaroo skin collagen peptide is in 1KDa hereinafter, the kangaroo skin collagen peptide is made by following steps:Kangaroo skin after unhairing and the degreasing of boiling in distilled water boiling water digests kangaroo skin using protease, obtains enzymolysis liquid;Enzymolysis liquid is centrifuged, takes supernatant freezing to be dried, obtains kangaroo skin collagen peptide.The kangaroo skin collagen peptide of the present invention can not only improve the added value of kangaroo production and processing, while can reduce environmental pollution, and obtain good Social benefit and economic benefit.The present invention kangaroo skin collagen peptide anti-oxidant, external moisturizing, whitening this three aspect have remarkable result, can be as antioxidant, moisturizer and the whitening agent in cosmetics.
Description
Technical field
This application involves biological product technical fields, more particularly to a kind of kangaroo skin collagen peptide and its as change
Application in cosmetic antioxidant, moisturizer and whitening agent.
Background technology
Collagen is the class protein that content is most abundant in the mammalian body, accounts for the 25- of internal total protein content
33%.Main constituents of the collagen as animal connective tissue, are primarily present in the tissues such as Animal Skin, bone, tendon,
Play holder effect and protective effect.The diversity and complexity of collagenic protein structure and function feature determine that it is being permitted
Multi-field critical role and good application prospect.
Collagen peptide is a kind of novel collagen class product, is to be with collagen or collagen-rich substance
What raw material was produced.It is that the product obtained after " hydrolysis " reaction occurs under certain external condition for collagen.It is so-called
External condition is exactly:The triple helix body of " collagen " this stabilization occurs in chemical action or under the action of bacterium, enzyme
Strand disintegrates, fracture, is passing through a kind of obtained product of processing.Both the peculiar amino acid with collagen formed for it,
Have the characteristics that molecular weight is small again.Therefore, it is easier through epidermis by dermal absorption, is also easy to be digested by alimentary canal,
There is good purposes in daily use chemicals, field of food health care.
About the preparation of collagen peptide, it is more common in is digested with the skin of fish, squama, bone or pig, the skin of ox and bone source at present
Processing obtains the research of collagen peptide.In addition, in the prior art, when extracting collagen from raw material, it usually needs right
The method extraction collagen of acidolysis or enzymolysis is first passed through, then the suitable protease of reselection digests collagen at small point
Sub- peptide, finally obtains collagen peptide.The molecular weight of collagen is big, is insoluble in water, is dissolved only in acid, the dissolving side of collagen
Formula is limited by very large.Therefore, the enzymolysis of collagen needs in acid condition, this largely affects enzyme
The selection of class, the requirement digested to pH is high, and only a small number of enzymes (such as the acid proteases such as pepsin) is available.Enzyme
The time of solution is long, and effect is poor, and yield is low, relatively complicated in extraction process.And using kangaroo skin as the collagen peptide of raw material also
Rare report.The collagenous fiber bundle of kangaroo skin is compared with the collagenous fiber bundle of general mammal skin, and type of weave is different, greatly
Part collagenous fiber bundle is parallel to surface and is woven in wave stratiform, is crossed-over between different levels, between layers
Alternate angle is less than 90 °, and the type of weave of each position collagenous fibres is essentially identical, and the elastomer of kangaroo skin is smaller, but is distributed ratio
It is more uniform.Due to its unique fibre structure, it is not only good leather articles for use and the potential resource of beauty product.Currently,
To the research of kangaroo skin also in kangaroo hide glue and leather product production technique, it is developed not completely.Therefore to kangaroo skin
Development and application will become focus and hot spot, while will produce huge economic benefit and social benefit.
Invention content
The present invention provides a kind of kangaroo skin collagen peptide, the average molecular weight of the kangaroo skin collagen peptide exists
1.0KDa following.
The present invention also provides a kind of preparation methods of kangaroo skin collagen peptide, include the following steps:
(1) kangaroo skin after unhairing and the degreasing of boiling in boiling water;
(2) it uses protease to carry out enzymolysis processing to the kangaroo skin after unhairing and degreasing, obtains enzymolysis liquid;
(3) enzymolysis liquid is centrifuged, supernatant is taken to be dried, obtain kangaroo skin collagen peptide.
The preparation method of above-mentioned kangaroo skin collagen peptide the step of in (1), time that kangaroo skin boils in boiling water
It it is 20-40 minutes, the dosage of boiling water is 10-30 times of volume of kangaroo skin weight (ml/g) after unhairing and degreasing.Water is with double steamings
Water is preferred, because distilled water impurity is less.It is of course also possible to use other clean water.
The preparation method of above-mentioned kangaroo skin collagen peptide the step of in (1), after the dosage of boiling water is unhairing and degreasing
Kangaroo skin weight 15-25 times of volume (ml/g).
The preparation method of above-mentioned kangaroo skin collagen peptide the step of in (2), the protease can be acidic protein
Any one of enzyme, alkali protease, neutral proteinase.Preferably, the protease be selected from papain, pepsin and
It is one or more in pancreatin, preferred papain.It is highly preferred that the weight ratio of the papain and the kangaroo skin
For 1%-5%, more preferably 2%.
Invention was attempted two kinds of enzymes being used in mixed way, and using ABTS free radical scavenging activities as index, by orthogonal experiment, obtained
The condition of best mixed enzyme.But since the time that mixed enzyme enzymolysis needs is long, process is cumbersome, the effect obtained and wood is applied alone
Melon protease is equally matched, therefore final only selection papain.Best enzymatic hydrolysis condition is had nothing in common with each other according to the type of enzyme.
The preparation method of above-mentioned kangaroo skin collagen peptide the step of in (2), when being digested using papain,
The condition of enzymolysis is:The weight ratio of papain and the kangaroo skin is 1-2%, and pH 5.4-9.4, temperature is 40-55 DEG C,
Enzymolysis time is 2-5 hours.Enzymolysis time cannot be too short, too short, and enzymolysis is not thorough.It is too long then not only need not, and to freedom
The removing of base will also result in certain negative effect.
The preparation method of above-mentioned kangaroo skin collagen peptide the step of in (3), the condition of the centrifugation is in room temperature ring
It is centrifuged 20-40 minutes with the rotating speed of 4000-5000rpm under border.
The preparation method of above-mentioned kangaroo skin collagen peptide the step of in (3), the drying includes freeze-drying and spray
Mist is dried, preferably freeze drying.
The invention also discloses above-described kangaroo skin collagen peptides to add as the antioxidant in cosmetics, whitening
Add agent and the application of moisturizer.
It is described anti-oxidant the invention also discloses a kind of antioxidant, whitening additive and moisturizer for cosmetics
Agent, whitening additive and moisturizer include above-described kangaroo skin collagen peptide.
It is described anti-oxidant the invention also discloses a kind of antioxidant, whitening additive and moisturizer for cosmetics
Agent, whitening additive and moisturizer include above-described kangaroo skin collagen peptide.
Kangaroo skin contains collagen, after the method processing appropriate of the biotechnology of modern zymetology, can obtain high-quality
Collagen peptide.The preparation method of kangaroo skin collagen peptide disclosed in the present application, processing raw material different from the past are answered
General labourer's sequence, and it has been to skip the technique that collagen is extracted from raw material, directly raw material is handled, it is good to obtain effect
High-quality collagen peptide, achieved the purpose that simplified preparation process.Kangaroo skin collagen peptide prepared by the application not only may be used
It to improve the added value of kangaroo production and processing, while can reduce environmental pollution, obtain good Social benefit and economic benefit.It is real
It tests and shows that the kangaroo skin collagen peptide of the application has remarkable result at anti-oxidant, external moisturizing, whitening this three aspect.With
People's HepG2 liver cancer cells are in the experiment that hydrogen peroxide models cell, and the kangaroo skin collagen peptide of the application is to cell antioxygen
Change ability increases, and has good effect to the removing of reactive oxygen species.The kangaroo skin collagen peptide of the application,
There is good inhibition to tyrosinase in vitro, and can effectively reduce the activity of tyrosinase in B16 melanocytes, says
It is bright that there is good white-skinned face function.
Description of the drawings
Fig. 1 is shown using ABTS free radical scavenging activities as index, is removed to ABTS under neutral proteinase difference enzymatic hydrolysis condition
The influence of ability;
Fig. 2 is shown using ABTS free radical scavenging activities as index, is removed to ABTS under acid protease difference enzymatic hydrolysis condition
The influence of ability;
Fig. 3 is shown using ABTS free radical scavenging activities as index, is removed to ABTS under alkali protease difference enzymatic hydrolysis condition
The influence of ability;
Fig. 4 is shown using ABTS free radical scavenging activities as index, is removed to ABTS under papain difference enzymatic hydrolysis condition
The influence of ability;
Fig. 5 is shown using ABTS free radical scavenging activities as index, and energy is removed to ABTS under pepsin difference enzymatic hydrolysis condition
The influence of power;
Fig. 6 is shown using ABTS free radical scavenging activities as index, and energy is removed to ABTS under trypsase difference enzymatic hydrolysis condition
The influence of power;
Fig. 7 shows the influence to yield and ABTS clearance rates under the different optimal enzymatic hydrolysis conditions of protease;Wherein, abscissa
For type (neutral proteinase, alkali protease, acid protease, papain, pepsin, the tryptose of protease
Enzyme), left and right ordinate is respectively polypeptide yield and ABTS clearance rates;
Fig. 8 shows the MALDI-TOF MS collection of illustrative plates of the kangaroo skin collagen peptide of the application;
Fig. 9 shows the ultraviolet absorption peak of the kangaroo skin collagen peptide of the application;
Figure 10 shows the moisture-retaining capacity of the kangaroo skin collagen peptide of the application, and the kangaroo skin in figure representated by ABC is more
Peptide be added concentration be respectively:A peptides (low) 10mg/ml;B peptides (in) 100mg/ml;C peptides (height) 1g/ml);
Figure 11 to Figure 13 shows suppression of the kangaroo skin collagen peptide prepared by the method for the present invention to external tyrosinase
It makes and uses;The kangaroo skin glue collagen polypeptide prepared by the method for the present invention plays inhibition to tyrosinase diphenolase and monophenolase,
It can not retroactive inhibition to being suppressed to for diphenolase;Figure 11 shows the kangaroo skin collagen peptide of the application to external tyrosinase two
The inhibition of phenolase, with the increase of peptide concentration, the vigor of enzyme continuously decreases;Figure 12 shows the kangaroo prepared by the method for the present invention
Inhibition type of the collagen peptide to external tyrosinase diphenolase, it can be seen that inhibiting type to be can not retroactive inhibition;Figure 13 is aobvious
Inhibiting effect of the kangaroo collagen peptide to external tyrosinase monophenolase of the application is shown, with the raising of peptide concentration, instead
The product of the time delay answered, reaction reduces;
Figure 14 shows the kangaroo skin collagen peptide prepared by the method for the present invention to increasing in Murine B 16 Melanoma Cells
Grow rate influence;Abscissa is kangaroo skin collagen peptide processing group (the concentration difference of kangaroo skin collagen peptide of various concentration
For 0,0.1,0.2,0.3,0.4,0.5 μ g/ μ L), ordinate is cell survival rate;
Figure 15 shows the kangaroo skin collagen peptide prepared by the method for the present invention to junket in Murine B 16 Melanoma Cells
The active influence of propylhomoserin;Abscissa be various concentration kangaroo skin collagen peptide processing group (kangaroo skin collagen peptide it is dense
Degree is respectively 0,0.1,0.2,0.3,0.4,0.5 μ g/ μ L), ordinate is the active relative activity of intracellular tyrosine;
Figure 16 shows that the ultra-oxygen anion free radical of the kangaroo skin collagen peptide prepared by the method for the present invention removes energy
Power;The kangaroo skin collagen peptide concentration that every curve represents is respectively (0,0.5,1,1.5 μ g/ μ L);
Figure 17 shows the kangaroo skin collagen peptide prepared by the method for the present invention to human hepatoma HepG2 cell's proliferation rate
It influences;Abscissa be various concentration kangaroo skin collagen peptide processing group (concentration of kangaroo skin collagen peptide is respectively 0,
0.1,0.2,0.3,0.4,0.5 μ g/ μ L), ordinate is cell opposite proliferation rate;
Figure 18 shows influence of the hydrogen peroxide to human hepatoma HepG2 cell's form;A, B, C, D, E, F are respectively A in figure:
Normal value (is not added with any medicament);B:100 μm of ol hydrogen peroxide;C:200 μm of ol/L hydrogen peroxide;D:300 μm of ol/L peroxidating
Hydrogen;E:400 μm of ol/L hydrogen peroxide;F:500 μm of ol/L hydrogen peroxide;
Figure 19 shows the kangaroo skin collagen peptide prepared by the method for the present invention to Hydroperoxide injury model cell shape
The influence of state;A, B, C, D, E, F are respectively in figure:A:Model group (600 μm of ol/L hydrogen peroxide);B:Normal group;C:600μ
+ 0.1 μ g/ μ L peptides of mol/L hydrogen peroxide;D:600 μm of+0.2 μ g/ μ L peptides of ol/L hydrogen peroxide;E:600 μm of ol/L hydrogen peroxide+
0.3 μ g/ μ L peptides;F:600 μm of+0.4 μ g/ μ L peptides of ol/L hydrogen peroxide);
Figure 20 to Figure 22 shows the kangaroo skin collagen peptide prepared by the method for the present invention to Hydroperoxide injury people liver
The influence of cancer HepG2 cell activity oxygen cluster contents (ROS);Experiment includes the control group (figure that dosage is respectively 0 μ g/ μ L
20), 600 μm of ol/L H2O2+ 0.50 μ g/ μ L kangaroo skins polypeptide Figure 21), 600 μm of ol/L H2O2(Figure 22);G-MEAN values are distinguished
For 105.6 (Figure 20), 118.94 (Figure 21), 221.3 (Figure 22).
Specific implementation mode
The condition research of neutral protease enzymolysis kangaroo skin
After kangaroo skin shaving, macroscopic white adipose is cut off with scissors, is cleaned, is drained, then take 10g kangaroos
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in skin.It waits for that it cools down room temperature, blends.Equivalent is divided into 5 parts.Every part
0.1g neutral proteinases (enzyme activity of the rich U.S. production in Hefei ,Anhui is 100u/mg), 50 DEG C of hydrolysis temperature, enzymolysis time 2h is added.
The pH value of enzymolysis is respectively 4.4,5.4,6.4,7.4,8.4.ABTS free radicals are removed as index using hydrolysate.As shown in Figure 1,
Illustrate for neutral proteinase, the active highest of kangaroo skin is digested when pH value 7.4, and hydrolysis result is best.
After kangaroo skin shaving, macroscopic white adipose is cut off with scissors, is cleaned, is drained, then take 10g kangaroos
15 times of volume distilled waters (150mL), electric rice cooker 40min is added in skin.It waits for that it cools down room temperature, blends.Equivalent is divided into 5 parts.Every part
0.1g neutral proteinases are added, the pH of enzymolysis is 7.4, enzymolysis time 2h.Hydrolysis temperature be 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55
℃.ABTS free radicals are removed as index using hydrolysate.As shown in Figure 1, illustrating for neutral proteinase, when temperature is 50 DEG C
The active highest of kangaroo skin is digested, hydrolysis result is best.
After kangaroo skin shaving, macroscopic white adipose is cut off with scissors, is cleaned, is drained, then take 10g kangaroos
25 times of volume distilled waters (250mL), electric rice cooker 30min is added in skin.It waits for that it cools down room temperature, blends.Equivalent is divided into 5 parts.Every part
Optimal pH 7.4 has been adjusted, 0.1g protease is added, is digested in 50 DEG C.Enzymolysis time is 2h, 3h, 4h, 5h, 6h.It is clear with hydrolysate
Except ABTS free radicals are index.As shown in Figure 1, explanation for neutral proteinase, digests the activity of kangaroo skin when the time is 5h
Highest, hydrolysis result are best.
After kangaroo skin shaving, macroscopic white adipose is cut off with scissors, is cleaned, is drained, then take 10g kangaroos
Skin is separately added into 10,15,20,25,30 times of volume distilled waters, electric rice cooker 30min.It waits for that it cools down room temperature, blends.Every part of tune
PH7.4, every part of addition 0.1g protease, 5h is digested at 50 DEG C, and ABTS free radicals are removed as index using hydrolysate.Such as Fig. 1 institutes
Show, illustrates for neutral proteinase, solid-liquid ratio 1:The active highest of kangaroo skin is digested when 10, hydrolysis result is best.
Acid protease digests the condition research of kangaroo skin
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.Every part is added 0.2g acid proteases and (wins the beautiful enzyme activity produced in Hefei ,Anhui
50u/mg), 45 DEG C of hydrolysis temperature, enzymolysis time 2h.The pH value of enzymolysis is respectively 2.4,3.4,4.4,5.4,6.4.To hydrolyze production
It is index that object, which removes ABTS free radicals,.As shown in Fig. 2, explanation, for acid protease, when pH value 3.4-4.4, digests kangaroo skin
Active highest, hydrolysis result is best.
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.The pH of every part of addition 0.2g acid protease, enzymolysis is 3.4, enzymolysis time 2h.
Hydrolysis temperature is 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C.ABTS free radicals are removed as index using hydrolysate.As shown in Fig. 2,
Illustrate for acid protease, the active highest of kangaroo skin is digested when temperature is 35 DEG C, and hydrolysis result is best.
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.Every part has been adjusted optimal pH 3.4, and 0.2g protease is added, is digested in 35 DEG C.Enzyme
It is 2h, 3h, 4h, 5h, 6h to solve the time.ABTS free radicals are removed as index using hydrolysate.As shown in Fig. 2, explanation is to acid egg
For white enzyme, the active highest of kangaroo skin is digested when enzymolysis time is 6h, hydrolysis result is best.
10g kangaroo skins (after shaving degreasing, drain) are separately added into 10,15,20,25,30 times of volume distilled waters, electric rice cooker
40min..It waits for that it cools down room temperature, blends.Every part has been adjusted optimal pH 3.4, and 0.2g protease is added, and (heavy weight hide with kangaroo skin is
2%) 6h, is digested in 35 DEG C, ABTS free radicals are removed as index using hydrolysate.As shown in Fig. 2, explanation is to acid protease
For, solid-liquid ratio 1:The active highest of kangaroo skin is digested when 20, hydrolysis result is best.
Alkali protease digests the condition research of kangaroo skin
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.Every part is added 0.1g alkali proteases and (wins the beautiful enzyme activity produced in Hefei ,Anhui
200u/mg), 40 DEG C of hydrolysis temperature, enzymolysis time 2h.The pH value of enzymolysis is respectively 8.4,9.4,10.4,11.4,12.4.With water
It is index to solve product and remove ABTS free radicals.As shown in figure 3, explanation, for alkali protease, when pH value 11.4, digests kangaroo
The active highest of skin, hydrolysis result are best.
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.The pH of every part of addition 0.1g alkali protease, enzymolysis is 11.4, enzymolysis time 2h.
Hydrolysis temperature is 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C.ABTS free radicals are removed as index using hydrolysate.As shown in figure 3,
Illustrate for alkali protease, the active highest of kangaroo skin is digested when temperature is 40 DEG C, and hydrolysis result is best.
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.The pH of every part of addition 0.1g alkali protease, enzymolysis is 11.4, the enzyme in 40 DEG C
Solution.Enzymolysis time is 2h, 3h, 4h, 5h, 6h.ABTS free radicals are removed as index using hydrolysate.The results are shown in Figure 3.Explanation
For alkali protease, enzymolysis time is the active highest that 4h digests kangaroo skin, and hydrolysis result is best.
10g kangaroo skins (after shaving degreasing, drain) are separately added into 10,15,20,25,30 times of volume distilled waters, electric rice cooker
20min.It waits for that it cools down room temperature, blends.Every part of addition 0.1g alkali protease, the pH of enzymolysis is 11.4, and 4h is digested in 40 DEG C,
ABTS free radicals are removed as index using hydrolysate.As shown in figure 3, illustrating for alkali protease, solid-liquid ratio 1:When 15
The active highest of kangaroo skin is digested, hydrolysis result is best.
The condition research of papain enzymolysis kangaroo skin
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.Every part is added 0.1g papains (enzyme activity of raw work production is 3500u/mg),
55 DEG C of hydrolysis temperature, enzymolysis time 2h.The pH value of enzymolysis is respectively 5.4,6.4,7.4,8.4,9.4.It is removed with hydrolysate
ABTS free radicals are index.As shown in figure 4, explanation is for papain, when pH value 7.4, the activity of enzymolysis kangaroo skin is most
Height, hydrolysis result are best.
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.The pH of every part of addition 0.3g papain, enzymolysis is 6.4, enzymolysis time 2h.
Hydrolysis temperature is 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C.ABTS free radicals are removed as index using hydrolysate.As shown in figure 4,
Illustrate for papain, the active highest of kangaroo skin is digested when temperature is 50 DEG C, and hydrolysis result is best.
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.Every part of addition 0.1g papain, the pH of enzymolysis are to be digested in 6.4,50 DEG C.
Enzymolysis time be 2h, 3h, 4h, 5h, 6h.ABTS free radicals are removed as index using hydrolysate.The results are shown in Figure 4, to pawpaw
For protease, enzymolysis 4h kangaroo skin hydrolysis results are best.
10g kangaroo skins (after shaving degreasing, drain) are separately added into 10,15,20,25,30 times of volume distilled waters, electric rice cooker
40min.It waits for that it cools down room temperature, blends.Every part of addition 0.1g papain, the pH of enzymolysis are to be digested in 6.4,50 DEG C, are digested
4h removes ABTS free radicals as index using hydrolysate.As shown in figure 4, illustrating for papain, solid-liquid ratio 1:20
When digest the active highest of kangaroo skin, hydrolysis result is best.
Pepsin digests the condition research of kangaroo skin
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.Every part is added 0.1g pepsins (enzyme activity of raw work production is 3500u/mg), enzyme
Solve 35 DEG C of temperature, enzymolysis time 2h.The pH value of enzymolysis is respectively 2.4,3.4,4.4,5.4,6.4.ABTS is removed with hydrolysate
Free radical is index.As shown in figure 5, explanation for pepsin, digests the active highest of kangaroo skin, enzymolysis when pH value 2.4
Effect is best.
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.The pH of every part of addition 0.4g pepsin, enzymolysis is 2.4, enzymolysis time 2h.Enzyme
It is 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C to solve temperature.ABTS free radicals are removed as index using hydrolysate.As shown in figure 5, saying
Bright that the active highest of kangaroo skin is digested when temperature is 35 DEG C for pepsin, hydrolysis result is best.
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.The pH of every part of addition 0.1g pepsin, enzymolysis is 2.4,35 DEG C of enzymolysis.Enzymolysis
Time is 2h, 3h, 4h, 5h, 6h.ABTS free radicals are removed as index using hydrolysate.As shown in figure 5, explanation is to pepsin
For, the active highest of kangaroo skin is digested when the time is 4h, hydrolysis result is best.
10g kangaroo skins (after shaving degreasing, drain) are separately added into 10,15,20,25,30 times of volume distilled waters, electric rice cooker
20min.It waits for that it cools down room temperature, blends.The pH of every part of addition 0.1g pepsin, enzymolysis is that 2.4,35 DEG C of enzymolysis digest 4h, with
It is index that hydrolysate, which removes ABTS free radicals,.As shown in figure 5, illustrating for pepsin, solid-liquid ratio 1:It is digested when 20
The active highest of kangaroo skin, hydrolysis result are best.
The condition research of trypsin digestion kangaroo skin
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.Every part is added 0.1g trypsase (enzyme activity of raw work production is 250u/mg), enzyme
Solve 35 DEG C of temperature, enzymolysis time 2h.The pH value of enzymolysis is respectively 5.4,6.4,7.4,8.4,9.4.ABTS is removed with hydrolysate
Free radical is index.As shown in fig. 6, explanation for trypsase, digests the active highest of kangaroo skin, enzymolysis when pH value 6.4
Effect is best.
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.The pH of every part of addition 0.1g trypsase, enzymolysis is 6.4, enzymolysis time 2h.Enzyme
It is 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C to solve temperature.ABTS free radicals are removed as index using hydrolysate.As shown in fig. 6, saying
Bright that the active highest of kangaroo skin is digested when temperature is 35 DEG C for trypsase, hydrolysis result is best.
20 times of volume distilled waters (200mL), electric rice cooker 20min is added in 10g kangaroo skins (after shaving degreasing, drain).It waits for
It cools down room temperature, blends.Equivalent is divided into 5 parts.Every part of addition 0.1g trypsase, the pH of enzymolysis are to be digested in 6.4,35 DEG C.Enzyme
It is 2h, 3h, 4h, 5h, 6h to solve the time.ABTS free radicals are removed as index using hydrolysate.As shown in fig. 6, explanation is to tryptose
For enzyme, the active highest of kangaroo skin is digested when the time is 4h, hydrolysis result is best.
10g kangaroo skins (degreasing, the kangaroo skin drained after shaving) are separately added into 10,15,20,25,30 times of volume distilled waters,
Electric rice cooker 20min.It waits for that it cools down room temperature, blends.The pH of every part of addition 0.5g trypsase, enzymolysis is enzyme in 6.4,35 DEG C
Solution digests 4h, and ABTS free radicals are removed as index using hydrolysate.As shown in fig. 6, illustrating for trypsase, solid-liquid ratio
It is 1:The active highest of kangaroo skin is digested when 20, hydrolysis result is best.
The condition optimizing of kangaroo skin collagen peptide extraction
Neutrality, acidity, alkalinity, pawpaw, trypsase optimum condition under, kangaroo skin polypeptide is extracted.
Using polypeptide yield and ABTS Scavenging activities as ordinate, enzyme class is mapped for abscissa.As a result such as Fig. 7 is shown, papain
Enzymolysis ability it is best.
Table 1 is the orthogonal experiment of papain enzymolysis kangaroo skin.Using ABTS clearance rates as measurement index, optimal extraction
Condition is that solid-liquid ratio is 1:20, pH 6.4,2% (140000u) enzyme amount is added, is hydrolyzed 3 hours at a temperature of 55 ± 0.5 DEG C
± 10 minutes.
Table 1
The average molecular weight of kangaroo skin collagen peptide measures
It is that solvent prepares 0.1%TFA (trifluoroacetic acid) with 50%CAN (acetonitrile), takes the molten Gly-His-Lys of 1ml ultrasounds weight.Draw 1 μ L
The molten peptide solution of weight and 1 μ L α-HCCA (alpha-cyano -4- hydroxycinnamic acids) matrix solution mixing point on mass spectrometer sample target, wait for
After sample substrate volatilization crystallization, upper machine is carried out with ground substance assistant laser analysis ionization time-of-flight mass spectrometer (MALDI-TOF MS)
Analysis.
The metallic element analysis of kangaroo skin collagen peptide
0.2g kangaroo skin collagen Gly-His-Lys are weighed, 8mL 65%-68% nitric acid is added and stands 30min.Micro-wave digestion 3 is small
Shi Hou stands cooling.It is settled to 250mL with distilled water, makes nitric acid ultimate density to 2%-3%.
ICP-MS (inductivity coupled plasma mass spectrometry) carries out metallic element analysis, and table 2 shows the kangaroo skin of the application
The content of collagen peptide metallic element.
Table 2
The amino acid analysis of kangaroo skin collagen peptide
Weigh 10g kangaroo skin collagen Gly-His-Lys, using the method for GB5009.124-2016, analysis of amino acid total amount and
The content of 16 kinds of amino acid.Table 3 shows the kangaroo skin collagen peptide total amino acid content of the application and containing for 16 kinds of amino acid
Amount.
Table 3
Embodiment 1
Take the kangaroo skin that 10g is drained, shaving.It is cut into the fritter of 1cm*1cm or so, 20 times of double steamings of volume (200ml) are added
Water, electric rice cooker 20min.It waits for that it cools down room temperature, blends.PH6.4 is adjusted, 2% papain is added, at 55 ± 0.5 DEG C
At a temperature of hydrolyze ± 10 minutes 3 hours.Heating, which is boiled 10 minutes, makes enzyme-deactivating.Again at room temperature, 5000rpm centrifuges 30 ± 5 points
Clock.Supernatant, lyophilization is taken to obtain kangaroo skin collagen Gly-His-Lys.
Fig. 8 shows the MALDI-TOF MS collection of illustrative plates for the kangaroo skin collagen peptide that embodiment 1 obtains.Embodiment 1 obtains
Kangaroo skin collagen peptide molecular weight in 1KDa or less.Molecular weight is smaller, is digested when illustrating using Papain enzymatic treatment
Fully.
Embodiment 2
Take the kangaroo skin that 10g is drained, shaving.It is cut into the fritter of 1cm*1cm or so, 20 times of double steamings of volume (200ml) are added
Water, electric rice cooker 20min.It waits for that it cools down room temperature, blends.PH6.4 is adjusted, 2% papain is added, at 55 DEG C ± 0.5 DEG C
At a temperature of hydrolyze ± 10 minutes 3 hours.Heating, which is boiled 10 minutes, makes enzyme-deactivating.Again at room temperature, 5000rpm centrifugations 30 ± 5
Minute.Supernatant, lyophilization is taken to obtain kangaroo skin collagen Gly-His-Lys.
Fig. 9 shows the aqueous solution of the kangaroo skin collagen peptide that example 2 obtains after ultraviolet full wavelength scanner in 220nm
There is maximum absorption band in left and right.This is the characteristic absorption peak of collagen.
Embodiment 3
The external moistening effect of the kangaroo skin collagen peptide of the application
According to the difference of moisturizer performance of keeping humidity, various concentration moisturizer is different to the active force of hydrone, absorbs moisture
It is also different with the ability of holding moisture.Sample containing certain moisture is placed in the drier of constant temperature and humidity to dry, timing weighing
The reduction of sample quality can compare the size of different sample moisture retentions by comparative analysis.For preferably simulated skin
Actual conditions, 3M adhesive plasters are sticked to glass slide, by each tested material (kangaroo skin peptide (low concentration) 10mg/ml;Peptide (middle concentration)
100mg/ml;Peptide (high concentration) 1g/ml) it applies thereon, it is put into and fills the dry of saturated ammonium sulfate solution (relative humidity 85%)
It in dry device, weighed every 2 hours, calculates the percentage of water loss of the period, the result is shown in Figure 12.Percentage of water loss calculation formula is:
Percentage of water loss (%)=(m1-m2)/m1 × 100%
Wherein m1 is the initial biodiversity of sample, and m2 is the biodiversity after sample is placed 2 hours.
The results show that in the kangaroo skin polypeptide of the application three, the peptide moistening effect of peptide (low) 10mg/ml is best.
Embodiment 4
The whitening effect of the kangaroo skin collagen peptide of the application
1, semi-inhibit rate of the kangaroo skin collagen peptide to external tyrosinase
Effect of the kangaroo skin collagen peptide to tyrosinase diphenolase is measured, its semi-inhibit rate is studied.It measures and uses 3mL
System.Effector is dissolved in water, and the solution of the effector containing various concentration is made.Using 0.5mmol/L L-DOPA as substrate,
In the 3mL live body systems of 0.05mol/L phosphate buffers (pH 6.8), the effector of 0.1mL various concentrations is first added in colorimetric
In cup, the substrate solution that 2.8mL is kept the temperature in 30 DEG C of waters bath with thermostatic control in advance is added, it is molten that 0.1mL Mushroom Tyrosinases are then added
Liquid, at once mixing measure the growth straight line of OD value that wavelength is 475nm at any time, from straight line under 30 DEG C of constant temperatures
Slope acquire enzyme activity.It is mapped with the relative surplus vigor pairing effect object concentration of enzyme, obtains the concentration-response curve of effector,
When by enzyme relative surplus vigor being 50%, corresponding obtained effector concentration acquires the IC50 values of effector.Figure 10 shows this
The kangaroo skin collagen peptide of application is 55mg/ml to the IC50 of tyrosinase.
2, inhibition type of the kangaroo skin collagen peptide to external tyrosinase
The IC50 values obtained according to above-mentioned steps.Change the amount for the zymoprotein being added, measures various concentration effector to mushroom
Mushroom tyrosinase catalysis L-DOPA (levodopa) aoxidizes the influence of vigor.With remaining enzyme activity pair of the enzyme after effector functions
The enzyme amount of addition is mapped, and thus judges Inhibitory Mechanism of the effector to Mushroom Tyrosinase diphenolase.Figure 13 shows this
The kangaroo skin collagen peptide of application is to the Inhibitory Mechanism of tyrosinase diphenolase can not retroactive inhibition.
3, kangaroo skin glue collagen polypeptide is to tyrosinase monophenolase activity analysis
The single phenol enzyme activity determination of tyrosinase measures effector to Mushroom Tyrosinase list using L-Tyrosine as substrate
The kinetic curve of phenol enzymic catalytic reaction influences.Using 3mL systems, with 2mmol/L (final concentration) Tyr (tyrosinase monophenolases
Substrate) it is that 0.1ml is first added in the live body system of 3mL 0.05mol/L phosphate buffers (pH 6.8) (final concentration) in substrate
Kangaroo skin glue collagen polypeptide containing various concentration is in cuvette, then adds 2.8ml0.05mol/L phosphate buffers, in advance
In the substrate solution of 30 DEG C of waters bath with thermostatic control heat preservation, 100 μ L of Mushroom Tyrosinase are then added, at once mixing, in 30 DEG C of constant temperature items
Measurement wavelength is the growth straight line of the OD value of 475nm at any time under part.As a result as shown in figure 13.
4, the influence that kangaroo skin collagen peptide is proliferated Murine B 16 Melanoma Cells
Figure 11 shows influence of the kangaroo skin collagen peptide of the application to being proliferated in Murine B 16 Melanoma Cells.
Using RPMI-1640 culture mediums (containing 10% newborn calf serum, penicillin 100U/mL, 100 μ g/mL of streptomysin), in CO2
37 DEG C of incubator, CO2Cell is cultivated under the conditions of=5% saturated humidity.Cell growth is waited for nearly Fusion Strain, through 0.25% pancreas egg
Cell concentration is collected and is adjusted in white enzymic digestion, and in 96 porocyte culture plates, it is unicellular outstanding that murine melanoma cells B16 is added
Liquid overnight, after adherent, abandons culture solution per 200 μ L of hole.The culture solution of the various concentration of polypeptide containing kangaroo skin is added simultaneously, continues
The 0.5mg/mL MTT (4,5- dimethylthiazole -2) (being protected from light) of 20 μ L are added after culture for 24 hours per hole, in CO237 DEG C of incubator, CO2
After cultivating cell 4h under the conditions of=5% saturated humidity, supernatant is abandoned, 180 μ L DMSO are added per hole and keep away at ambient temperature
Light shakes 10min or so, and first is made to be completely dissolved for crystallization, measures 570nm absorbance values in microplate reader immediately.Each concentration sets 5
A multiple holes, are averaged.It tests each time and takes same passage cell.
Abscissa is that (concentration of kangaroo skin collagen peptide is respectively for the kangaroo skin collagen peptide processing group of various concentration
0,0.1,0.2,0.3,0.4,0.5 μ g/ μ L), ordinate is cell opposite proliferation rate.Figure 14 is illustrated in 0~0.5 μ g/ μ L models
In enclosing, kangaroo skin collagen peptide does not have toxic action to Murine B 16 Melanoma Cells.
5, influence of the kangaroo skin collagen peptide to tyrosine activity in Murine B 16 Melanoma Cells
Cell culture processes are same as above.Cell growth is waited for nearly Fusion Strain, 0.25% trypsin digestion is collected and adjusted
Cell concentration, 2 × 104Be inoculated in 6 orifice plates, overnight, liquid changed after adherent, be separately added into containing 0,0.1,0.2,0.3,0.4,
The culture medium 2mL of the kangaroo skin collagen peptide of 0.5 μ g/ μ L.Culture solution is abandoned after continuing culture 24 hours.With the PBS of pH 7.4
Buffer solution washs 2 times, and the 900 μ L of PBS buffer solution that volume fraction is 1%TritonX-100 are added per hole, 100 μ L are then added
1.0mg/mL L-DOPA, ultrasound 2 minutes, are then cultivated 30 minutes at 30 DEG C, measure the absorbance value at 475nm.Each concentration
Processing sets 3 repetitions, is averaged.Experiment takes same passage cell every time.
Figure 12 shows the kangaroo skin collagen peptide of the application to tyrosine activity in Murine B 16 Melanoma Cells
It influences.Under the action of concentration range is 0~0.5 μ g/ μ L kangaroo skin collagen peptides, in Murine B 16 Melanoma Cells
Tyrosine activity shows the trend of reduction with the raising of kangaroo skin collagen peptide concentration.
Embodiment 5
The oxidation resistance of the kangaroo skin collagen peptide of the application
1, Scavenging activity of the kangaroo skin collagen peptide to ultra-oxygen anion free radical
Kangaroo skin collagen peptide to the Scavenging activity of ultra-oxygen anion free radical by mouse thymus cells rate come
It weighs.Tris-HCl buffer solutions (50mmol/L, pH 8.2,4.5mL) are mixed with pure water (4.2mL), 25 DEG C of incubation 20min, soon
Speed records 5min after 0.3mL pyrogallols solution (3mmol/L is prepared with 10mmol/L hydrochloric acid, through 25 DEG C of preheatings) mixing is added
Absorbance change of the interior reaction solution at 320nm draws time (t)-absorbance (A) curve, is calculated in the range of linearity anti-
The change for answering unit interval internal absorbance obtains the autoxidation rate of pyrogallol.
The measurement of the lower mouse thymus cells rate of sample to be tested effect:Tris-HCl buffer solutions (50mmol/L, pH
8.2,4.5mL) it is mixed with pure water (3.3mL), 25 DEG C of incubation 20min rapidly join 0.9mL samples and 0.3mL pyrogallols are molten
Liquid (3mmol/L is prepared with 10mmol/L hydrochloric acid, through 25 DEG C of preheatings) after mixing in record 5min reaction solution at 320nm
Absorbance change draws time (t)-absorbance (A) curve, and changing for reacton time internal absorbance is calculated in the range of linearity
Become, obtains the autoxidation rate of the lower pyrogallol of tested material effect.
Ultra-oxygen anion free radical clearance rate (%)=(1-V1/V0) * 100%
V0:Mouse thymus cells rate
V1:For the autoxidation rate of pyrogallol under sample to be tested
As shown in Figure 13 under the effect of kangaroo skin collagen peptide, with the increase of concentration, the autoxidation speed of pyrogallol
Rate reduces, and ultra-oxygen anion free radical clearance rate increases.Illustrate that kangaroo skin collagen peptide has good radical scavenging activity
Power.
2, Scavenging activity of the kangaroo skin collagen peptide to hydroxyl radical free radical
It is competed by salicylic acid and captures hydroxyl radical free radical method, to measure kangaroo skin collagen peptide to the clear of hydroxyl radical free radical
Removing solid capacity.The kangaroo skin collagen peptide (0.25,0.5,0.75,1,1.5 μ g/ μ L) of 1mL various concentrations and 1mL ferrous sulfate
(FeSO4, 9mmol/L) and 1mL hydrogen peroxide (10mmol/L) mixing, 37 DEG C of incubation 10min, addition 1mL salicylic acids (9mmol/
L), 37 DEG C of incubation 10min after mixing, measure the absorbance of reaction solution, pure water makees blank control at 510nm.It counts as the following formula
Calculate sample to be tested OH-Free radical scavenging activity.The results are shown in Table 4.
Hydroxyl radical free radical clearance rate (%)=(1-A experimental groups light absorption value/A blank groups light absorption value) * 100%
Table 4
| Concentration | 0.25μg/μL | 0.5μg/μL | 0.75μg/μL | 1μg/μL | 1.5μg/μL |
| Clearance rate | 33% | 44.75% | 58% | 70% | 95% |
3, Scavenging activity of the kangaroo skin collagen peptide to DPPH
DPPH methods take the kangaroo collagen egg of 0.1ml various concentrations according to the established method such as Brand-Williams
White peptide (0,2.5,5,7.5,10 μ g/ μ L), is added 3ml 0.004%DPPH methanol solutions.After mixing, after standing 30min
Light absorption value is measured at 517nm.Blank sample uses methanol.The results are shown in Table 5.
Inhibiting rate (%)=[(A0-A1)/A0]* 100%
Wherein A0(adding 0.1mL methanol and 3mL DPPH methanol solutions) is the light absorption value being added before sample, A1For sample is added
Light absorption value afterwards.
Table 5
| Concentration | 0μg/μL | 2.5μg/μL | 5μg/μL | 7.5μg/μL | 10μg/μL |
| Clearance rate | 0 | 13% | 23% | 28% | 34% |
4, Scavenging activity of the kangaroo skin collagen peptide to ABTS free radicals
Early-stage preparations:ABTS 96mg are taken, distilled water 25mL is added to be made into A liquid;Take K2S2O3784mg adds distilled water 10mL to match
At B liquid.By 5ml A liquid and 88 μ L B liquid mixings, 12-16 hours are stood, ABTS working solutions are configured to.
ABTS working solutions are diluted with PBS solution, it is desirable that light absorption value is 0.7 ± 0.02 at 734nm at normal temperatures.
0.2mLABTS working solutions mix, room temperature with the kangaroo skin collagen peptide (0,2.5,5,7.5,10 μ g/ μ L) of 10uL various concentrations
It is protected from light and stands 6min, absorbance is surveyed at 734nm wavelength.The results are shown in Table 6.
ABTS free radical scavenging activities (100%)=(A0-Ai/A0) * 100%
Wherein A0To be not added with sample, the absorbance of ABTS is added;Ai is the absorbance that sample and ABTS is added
Table 6
| Concentration | 0μg/μL | 2.5μg/μL | 5μg/μL | 7.5μg/μL | 10μg/μL |
| Clearance rate | 0% | 15% | 32% | 51% | 60% |
Embodiment 6
Application of the kangaroo skin glue collagen polypeptide of the application in anti-oxidant model
1, influence of the kangaroo skin collagen peptide to human hepatoma HepG2 cell's proliferation rate.
Figure 17 shows influence of the kangaroo skin collagen peptide of the application to human hepatoma HepG2 cell's proliferation rate.Using
DMEM culture mediums (contain 10% newborn calf serum, penicillin 100U/mL, 100 μ g/mL of streptomysin), in CO237 DEG C of incubator,
CO2Cell is cultivated under the conditions of=5% saturated humidity.Cell growth is waited for nearly Fusion Strain, through 0.25% trypsin digestion
Cell concentration is collected and adjusted, in 96 porocyte culture plates, human hepatoma HepG2 cell's single cell suspension is added, per 200 μ of hole
L after adherent, abandons culture solution overnight.The culture solution of the various concentration of polypeptide containing kangaroo skin is added simultaneously, it is rear for 24 hours every to continue culture
The 0.5mg/mL MTT (being protected from light) of 20 μ L are added in hole, in CO237 DEG C of incubator, CO2Cell is cultivated under the conditions of=5% saturated humidity
After 4h, supernatant is abandoned, 180 μ L DMSO are added per hole, at ambient temperature, concussion 10min or so is protected from light, makes first for having crystallized
Fully dissolved measures 570nm absorbance values in microplate reader immediately.Each concentration sets 5 multiple holes, is averaged.Experiment is equal each time
Take same passage cell.
Abscissa is that (concentration of kangaroo skin collagen peptide is respectively for the kangaroo skin collagen peptide processing group of various concentration
0,0.1,0.2,0.3,0.4,0.5 μ g/ μ L), ordinate is cell opposite proliferation rate.Figure 17 is illustrated in 0~0.5 μ g/ μ L models
In enclosing, kangaroo skin collagen peptide does not have toxic action to human hepatoma HepG2 cell, and to human liver cancer in this concentration range
HepG2 cell Proliferations do not influence.
2, the foundation of Hydroperoxide injury human hepatoma HepG2 cell model
The HepG2 cells in exponential phase are taken, are inoculated in 6 orifice plates, the culture in 37 DEG C, 5%CO2 (v/v)
12h, discards old culture solution, after the hydrogen peroxide treatment HepG2 cells of various concentration (0~500 μM) are added for 24 hours, Jim Sa dye
After the processing of color liquid, 6 orifice plates are placed on inverted phase contrast microscope, the form of different groups of cells is observed, and are taken pictures.As a result such as Figure 18
Shown, control group HepG2 cells, cell space is full, the smooth of the edge, clear-cut, and fusiformis or irregular polygon is presented.Peroxidating
In hydrogen processing group, cell quantity is reduced, and is distributed lax, a small amount of cell rounding.
3, influence of the kangaroo skin collagen peptide to Hydroperoxide injury model cell form
Logarithmic growth phase HepG2 cells, are inoculated in 6 orifice plates, in 37 DEG C, 5%CO2(v/v) culture for 24 hours, discards in
Old culture solution discards culture solution after the drug-treated HepG2 cells of various concentration are added for 24 hours.PBS is washed 2 times, and methanol is fixed
15min, PBS are washed 2 times, and 500 μ L Hochest dyeing liquors are added per hole, are protected from light and are incubated 10min, discard dyeing liquor, be placed in glimmering
The form of nucleus is observed under light inverted microscope, and is taken pictures.As a result as shown in figure 19, normal to organize HepG2 cells, cell space is full
Full, the smooth of the edge is clear-cut, and fusiformis or irregular polygon is presented.In hydrogen peroxide treatment group, cell quantity is reduced, point
Cloth is lax, and downright bad cell is more.The cell that hydrogen peroxide and kangaroo skin polypeptide are handled together, with the raising of peptide concentration,
Cell quantity gradually increases, and downright bad quantity gradually decreases.
4, kangaroo skin collagen peptide is to reactive oxygen species (ROS) content in Hydroperoxide injury human hepatoma HepG2 cell
It influences
The HepG2 cells of logarithmic growth phase, are inoculated in 6 orifice plates, in 37 DEG C, 5%CO2(v/v) culture for 24 hours, is abandoned in
Old culture solution is removed, after the drug-treated HepG2 cells of various concentration are added for 24 hours, discards culture solution.It is collected with trypsin digestion
Cell is in the Ep pipes of 1.5mL.It is primary to clean remaining cell with 1mL PBS again, is also collected into same Ep pipes, 2500rpm from
The heart 5 minutes goes after supernatant to use 1mL PBS that cell is resuspended again, and supernatant is removed in centrifugation again.After cell is resuspended in 1mL PBS, it is added
The 1 μ L of probe DCFH-DA (dichlorofluorescein diacetate esters) of 10mmol/L are placed in 37 DEG C and are incubated 30 minutes.Then centrifuging and taking again
Cell is resuspended with 1mL PBS in supernatant.The analysis of ROS is carried out to cell on flow cytometer.
Figure 20 shows the kangaroo skin collagen peptide of the application to Hydroperoxide injury human liver cancer cell HepG2 active oxygens
The influence of cluster content.From left to right it is followed successively by the control group of 0 μ g/ μ L, 600 μm of ol/LH2O2And 0.50 μ g/ μ L kangaroo collagen eggs
White polypeptide, 600 μm of ol/LH202.G-MEAN values are respectively from left to right 105.6,118.94,221.3.
G-MEAN values are bigger, illustrate that ROS activity is higher.In hydrogen peroxide and kangaroo skin collagen peptide existing feelings together
Under condition, although the activity of ROS is higher than control group, inferior to processing group existing for only hydrogen peroxide.Illustrate kangaroo collagen egg
White peptide has Scavenging activity to reactive oxygen species, can reduce the oxidative damage of cell.
Therefore, the kangaroo skin collagen peptide of the application has good anti-oxidation efficacy.
In conclusion these are only the preferred embodiment of the application, it is not intended to limit the protection domain of the application.
Therefore, any modification, equivalent replacement, improvement and so within the spirit and principles of this application, should be included in this Shen
Within protection domain please.
Claims (9)
1. a kind of kangaroo skin collagen peptide, which is characterized in that the average molecular weight of the kangaroo skin collagen peptide is in 1KDa
Below.
2. kangaroo skin collagen peptide as described in claim 1, which is characterized in that the kangaroo skin collagen peptide by with
Lower step is made:
(1) boil the kangaroo skin after degreasing and unhairing in distilled water boiling water;Wherein, the time boiled in boiling water is 20-40
Minute, based on ml/g, the dosage of boiling water is 10-30 times of volume of the kangaroo skin weight after degreasing and unhairing;
(2) it uses protease to carry out enzymolysis processing to the kangaroo skin after unhairing and degreasing, obtains enzymolysis liquid;
(3) enzymolysis liquid is centrifuged, supernatant is taken to be freeze-dried or be spray-dried, obtain kangaroo collagen
Peptide.
3. kangaroo skin collagen peptide as described in claim 1, which is characterized in that in step (2), the protease is wood
The condition of melon protease, enzymolysis is:The weight ratio of papain and the kangaroo skin is 1-2%, pH 5.4-9.4, temperature
It it is 40-55 DEG C, enzymolysis time is 2-5 hours.
4. a kind of antioxidant for cosmetics, which is characterized in that the antioxidant includes as claimed in claim 1 or 2
Kangaroo skin collagen peptide.
5. a kind of whitening additive for cosmetics, which is characterized in that the whitening additive includes claims 1 or 2 institute
The kangaroo skin collagen peptide stated.
6. a kind of moisturizer for cosmetics, which is characterized in that the moisturizer includes kangaroo as claimed in claim 1 or 2
Collagen peptide.
7. kangaroo skin collagen peptide as claimed in claim 1 or 2 is as the application in the antioxidant in cosmetics.
8. kangaroo skin collagen peptide as claimed in claim 1 or 2 is as the application in the whitening additive in cosmetics.
9. kangaroo skin collagen peptide as claimed in claim 1 or 2 is as the application in the moisturizer in cosmetics.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1653087A (en) * | 2002-05-21 | 2005-08-10 | 科尔泰克澳大利亚有限公司 | Collagen and method for producing same |
| US20110020864A1 (en) * | 2009-07-27 | 2011-01-27 | National Cheng Kung University | Preparation of High Purity Collagen |
| CN102559826A (en) * | 2012-02-15 | 2012-07-11 | 胡如桂 | Method for preparing collagen oligopeptide |
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2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1653087A (en) * | 2002-05-21 | 2005-08-10 | 科尔泰克澳大利亚有限公司 | Collagen and method for producing same |
| US20110020864A1 (en) * | 2009-07-27 | 2011-01-27 | National Cheng Kung University | Preparation of High Purity Collagen |
| CN102559826A (en) * | 2012-02-15 | 2012-07-11 | 胡如桂 | Method for preparing collagen oligopeptide |
Non-Patent Citations (2)
| Title |
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| ELZBIETA GURDAK,ET AL.: "Factors and mechanisms determining the formation of fibrillar collagen structures in adsorbed phases", 《COLLOIDS AND SURFACES B: BIOINTERFACES》 * |
| 涂颖科等: "袋鼠皮加工技术", 《中国皮革》 * |
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