CN104593458A - Rana japonica oil antioxidant polypeptide - Google Patents
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Abstract
The invention relates to a rana japonica oil antioxidant polypeptide. The rana japonica oil contains abundant nutrient substance, however in the existing using method of rana japonica oil, the nutrient substance is not likely to be absorbed by a human body due to too big molecular weight of proteins, the polypeptide with even molecular weight still cannot be obtained after rana japonica oil is subjected to enzymolysis by a conventional method, the efficient utilization of nutrient substances cannot be realized. The invention provides a rana japonica oil polypeptide with small molecular weight, which is prepared by the method specifically comprising the following steps: adjusting the pH (potential of hydrogen) of a rana japonica oil solution to 1.6-1.8, adding 0.1-0.3g/ml pepsase to carry out enzymolysis completely; adjusting the pH of the solution to 6.0-7.0, adding 0.2-0.4g/ml neutral protease to carry out enzymolysis completely; adjusting the pH of the solution to 7.0-8.0, adding bromelain to carry out enzymolysis completely; heating to 70-75 DEG C, inactivating the enzyme to obtain enzymolysis solution; filtering with a 0.3-0.4mu m filter film, and then filtering with a ultra-filtration film with molecular weight cut-off of 4-5kD, adding activated carbon, stirring, filtering, concentrating and drying, so as to obtain rana japonica oil polypeptide; the polypeptide can be easily absorbed and has an excellent antioxidant effect.
Description
Technical field
The present invention relates to protein polypeptide field, be specifically related to a kind of wood frog oil antioxidation polypeptide.
Background technology
Wood frog oil, is commonly called as Oviductus Ranae or wood frog's fallopian tube cream, and Compendium of Material Medica is recorded: Oviductus Ranae, is referred to as in Compendium of Material Medica " Kazakhstan, mountain ", another name Oviductus Ranae, wood frog's fallopian tube.Be named as Rana temporaria chensinensis David now.Primary growth is in mountain area, Northeast China, comprise Changbai Mountain arteries and veins, large portion, the Xiaoxinanlin Mountains, innerland, Zhang Guangcai ridge, pure wildlife, is also called as Chinese forest frog (Oviductus Ranae), it is the distinctive frog kind in the Northeast, the dry thing of its fallopian tube of female frog is referred to as " snow frog oil ", the effect have qi-restoratives moistening lung, improving the health, and is just described as the handed down from ancient times excellent tonic product equally celebrated for their achievements with northeast Triratna among the people from ancient times.Widely people's application over the past thousands of years, its pharmacological action is also known already.Wood frog oil is the uterine tube of female frog bosom maturation of ovum phase, it contains the biologically active factorss such as a large amount of amino acid, inorganic elements, VITAMIN and multiple complex polypeptide, be rich in three kinds of sexual hormoue especially, i.e. estradiol, testosterone, progesterone, there is the title medically having " soft gold ", there is the strong kidney of significant enriching yin and excite immunologic function and regulation mechanism effect, be widely used in the fields such as food, healthcare products and makeup.
At present, take wood frog oil as the skin-protection product of Raw material processing, the molecular weight due to albumen is excessive and be not easily absorbed by the body.After adopting a conventional step enzymolysis process to carry out enzymolysis, the polypeptide molecular weight heterogeneity obtained, and the content of peptides of small-molecular-weight is few, still cannot realize the efficiency utilization of nutritive substance.Therefore, need badly a kind of that can be absorbed by the body, that antioxidant effect is good Rana oil polypeptide is provided.
Summary of the invention
Object of the present invention is the defect overcoming prior art, and provide a kind of antioxidation polypeptide taking wood frog oil as raw material and extract, described in this, the molecular weight of polypeptide is little, and has excellent anti-oxidant activity, is with a wide range of applications.
The invention provides a kind of wood frog oil antioxidation polypeptide, described antioxidation polypeptide take wood frog oil as raw material, is prepared from by the method comprised the following steps:
1) get wood frog oil, remove manadesma, add 2 ~ 10 times to the water of wood frog oil volume, be placed in 2 ~ 4 DEG C of bubbles and send out 8 ~ 12h, obtain wood frog oil solution;
2) regulating step 1) pH value of gained wood frog oil solution is 1.6 ~ 1.8, add 0.1 ~ 0.3g/ml stomach en-, at 35 ~ 40 DEG C, abundant enzymolysis, obtains Pepsin enzymolysis solution;
3) regulating step 2) pH value of gained Pepsin enzymolysis solution is 6.0 ~ 7.0, add 0.2 ~ 0.4g/ml neutral protease, at 45 ~ 55 DEG C, abundant enzymolysis, obtains neutral protein enzymolysis liquid;
4) regulating step 3) pH value of gained neutral protease enzymolysis liquid is 7.0 ~ 8.0, add 0.2 ~ 0.4g/ml bromeline, at 55 ~ 60 DEG C, abundant enzymolysis, obtains pineapple enzymolysis solution;
5) by step 4) gained pineapple enzymolysis solution is heated to 70 ~ 75 DEG C, and leave standstill 10 ~ 15min, make enzyme-deactivating, obtain enzymolysis solution;
6) step 5 is got) gained enzymolysis solution, use the membrane filtration of 0.3 ~ 0.4 μm, collect filtrate, re-use the ultrafiltration membrance filter that molecular weight cut-off is 4 ~ 5kD, collect filtrate, after adding gac room temperature stirring at low speed 1 ~ 3h, cross and filter gac, vacuum concentration, spraying dry, obtains wood frog oil antioxidation polypeptide.
The wood frog oil that the present invention uses can be commercially available wood frog oil, is preferably fresh wood frog oil.In order to make the albumen in woods silicone oil fully resolve into micromolecule polypeptide, the present invention adopts the multistep enzymolysis of stomach en--neutral protease-bromeline, specifically:
Step 2 of the present invention) in, described pepsic specific activity is 3000 ~ 5000U/g, and add-on is preferably 0.2g/ml, and hydrolysis temperature is preferably 38 DEG C; Enzymolysis should be abundant, if but overlong time, can have an impact to polypeptide active, enzymolysis time is preferably 4 ~ 5h, more preferably 4.5h;
Step 3 of the present invention) in, the specific activity of described neutral protease is 40000 ~ 70000U/g, and add-on is preferably 0.3g/ml, and hydrolysis temperature is preferably 50 DEG C; Enzymolysis should be abundant, if but overlong time, can have an impact to polypeptide active, enzymolysis time is preferably 3 ~ 4h, more preferably 3.5h;
Step 4 of the present invention) in, the specific activity of described bromeline is 600000 ~ 900000U/g, and add-on is preferably 0.3g/ml, and hydrolysis temperature is preferably 58 DEG C; Enzymolysis should be abundant, if but overlong time, can have an impact to polypeptide active, enzymolysis time is preferably 2 ~ 3h, more preferably 2.5h.
Preferably, wood frog oil antioxidation polypeptide of the present invention is prepared from by the method comprised the following steps:
1) get wood frog oil, remove manadesma, add 2 ~ 10 times to the water of wood frog oil volume, be placed in 2 ~ 4 DEG C of bubbles and send out 8 ~ 12h, obtain wood frog oil solution;
2) regulating step 1) pH value of gained wood frog oil solution is 1.6 ~ 1.8, the specific activity adding enzyme is 0.1 ~ 0.3g/ml stomach en-of 3000 ~ 5000U/g, and enzymolysis 4 ~ 5h at 35 ~ 40 DEG C, obtains Pepsin enzymolysis solution;
3) regulating step 2) pH value of gained Pepsin enzymolysis solution is 6.0 ~ 7.0, the specific activity adding enzyme is 0.2 ~ 0.4g/ml neutral protease of 40000 ~ 70000U/g, and enzymolysis 3 ~ 4h at 45 ~ 55 DEG C obtains neutral protein enzymolysis liquid;
4) regulating step 3) pH value of gained neutral protease enzymolysis liquid is 7.0 ~ 8.0, the specific activity adding enzyme is 0.2 ~ 0.4g/ml bromeline of 600000 ~ 900000U/g, and enzymolysis 2 ~ 3h at 55 ~ 60 DEG C, obtains pineapple enzymolysis solution;
5) by step 4) gained pineapple enzymolysis solution is heated to 70 ~ 75 DEG C, and leave standstill 10 ~ 15min, make enzyme-deactivating, obtain enzymolysis solution;
6) step 5 is got) gained enzymolysis solution, use the membrane filtration of 0.3 ~ 0.4 μm, collect filtrate, re-use the ultrafiltration membrance filter that molecular weight cut-off is 4 ~ 5kD, collect filtrate, after adding gac room temperature stirring at low speed 1 ~ 3h, cross and filter gac, vacuum concentration, spraying dry, obtains wood frog oil antioxidation polypeptide.
In order to ensure the activity of the wood frog oil antioxidation polypeptide prepared, except enzyme digestion reaction is to except the special requirement of temperature, the service temperature of each step of the present invention is all preferably 0 ~ 4 DEG C.
The present invention protects described wood frog oil antioxidation polypeptide preparing the application in makeup, food, healthcare products, medicine further.
Compared with prior art, technical scheme provided by the invention overcomes the large or heterogeneity of albumen in wood frog oil, polypeptide molecular weight, thus causes the defect that is difficult to be absorbed by the body.In Rana oil polypeptide provided by the invention, contain abundant, that molecular weight is homogeneous micromolecule polypeptide, after testing, described wood frog oil antioxidation polypeptide has good antioxidant effect.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
Wood frog oil antioxidation polypeptide is prepared according to following steps:
1) get wood frog oil 200ml, remove manadesma, add 1600ml water, be placed in 3 DEG C of bubbles and send out 10h, obtain wood frog oil solution;
2) regulating step 1) pH value of gained wood frog oil solution is 1.7, the specific activity adding enzyme is the 0.2g/ml stomach en-of 4000U/g, and enzymolysis 4.5h at 38 DEG C, obtains Pepsin enzymolysis solution;
3) regulating step 2) pH value of gained Pepsin enzymolysis solution is 6.5, the specific activity adding enzyme is the 0.3g/ml neutral protease of 60000U/g, and enzymolysis 3.5h at 50 DEG C obtains neutral protein enzymolysis liquid;
4) regulating step 3) pH value of gained neutral protease enzymolysis liquid is 7.5, the specific activity adding enzyme is the 0.3g/ml bromeline of 800000U/g, and enzymolysis 2.5h at 58 DEG C, obtains pineapple enzymolysis solution;
5) by step 4) gained pineapple enzymolysis solution is heated to 72 DEG C, and leave standstill 12min, make enzyme-deactivating, obtain enzymolysis solution;
6) step 5 is got) gained enzymolysis solution, use the membrane filtration of 0.35 μm, collect filtrate, re-use the ultrafiltration membrance filter that molecular weight cut-off is 4kD, collect filtrate, after adding gac room temperature stirring at low speed 2h, cross and filter gac, vacuum concentration, spraying dry, obtains wood frog oil antioxidation polypeptide.
Embodiment 2
Wood frog oil antioxidation polypeptide is prepared according to following steps:
1) get wood frog oil 200ml, remove manadesma, add 400ml water, be placed in 2 DEG C of bubbles and send out 8h, obtain wood frog oil solution;
2) regulating step 1) pH value of gained wood frog oil solution is 1.6, the specific activity adding enzyme is the 0.1g/ml stomach en-of 3000U/g, and enzymolysis 4h at 35 DEG C, obtains Pepsin enzymolysis solution;
3) regulating step 2) pH value of gained Pepsin enzymolysis solution is 6.0, the specific activity adding enzyme is the 0.2g/ml neutral protease of 40000U/g, and enzymolysis 3h at 45 DEG C obtains neutral protein enzymolysis liquid;
4) regulating step 3) pH value of gained neutral protease enzymolysis liquid is 7.0, the specific activity adding enzyme is the 0.2g/ml bromeline of 600000U/g, and enzymolysis 2h at 55 DEG C, obtains pineapple enzymolysis solution;
5) by step 4) gained pineapple enzymolysis solution is heated to 70 DEG C, and leave standstill 10min, make enzyme-deactivating, obtain enzymolysis solution;
6) step 5 is got) gained enzymolysis solution, use the membrane filtration of 0.3 μm, collect filtrate, re-use the ultrafiltration membrance filter that molecular weight cut-off is 4kD, collect filtrate, after adding gac room temperature stirring at low speed 1h, cross and filter gac, vacuum concentration, spraying dry, obtains wood frog oil antioxidation polypeptide.
Embodiment 3
Wood frog oil antioxidation polypeptide is prepared according to following steps:
1) get wood frog oil 200ml, remove manadesma, add 2000ml water, be placed in 4 DEG C of bubbles and send out 12h, obtain wood frog oil solution;
2) regulating step 1) pH value of gained wood frog oil solution is 1.8, the specific activity adding enzyme is the 0.3g/ml stomach en-of 5000U/g, and enzymolysis 5h at 40 DEG C, obtains Pepsin enzymolysis solution;
3) regulating step 2) pH value of gained Pepsin enzymolysis solution is 7.0, the specific activity adding enzyme is the 0.4g/ml neutral protease of 70000U/g, and enzymolysis 4h at 55 DEG C obtains neutral protein enzymolysis liquid;
4) regulating step 3) pH value of gained neutral protease enzymolysis liquid is 8.0, the specific activity adding enzyme is the 0.4g/ml bromeline of 900000U/g, and enzymolysis 3h at 60 DEG C, obtains pineapple enzymolysis solution;
5) by step 4) gained pineapple enzymolysis solution is heated to 75 DEG C, and leave standstill 15min, make enzyme-deactivating, obtain enzymolysis solution;
6) step 5 is got) gained enzymolysis solution, use the membrane filtration of 0.4 μm, collect filtrate, re-use the ultrafiltration membrance filter that molecular weight cut-off is 5kD, collect filtrate, after adding gac room temperature stirring at low speed 3h, cross and filter gac, vacuum concentration, spraying dry, obtains wood frog oil antioxidation polypeptide.
Comparative example 1
Compared with embodiment 1, difference be only, carry out step 2) pepsin hydrolysis, step 1) after be step 3).
Comparative example 2
Compared with embodiment 1, difference be only, carry out step 3) neutral white enzymic hydrolysis, step 2) after be step 4).
Comparative example 3
Compared with embodiment 1, difference be only, carry out step 4) bromelain hydrolyzate, step 3) after be step 5).
Experimental example 1
1, sample: each embodiment and comparative example gained polypeptide, be configured to the solution that concentration is 5mg/ml respectively.
2, reference material: reductive glutathione (307Da), oxytocin (1007Da), hyperglycemic-glycogenolytic factor (3485Da) and Regular Insulin (5808Da), be configured to the standard substance substance solution of 2mg/ml respectively.
3, detection method: with the Tris-HCl elutriant Balance Treatment SPhedexG-25 gel column of 0.1mol/L, under the wavelength of 220nm, examination criteria substance solution, according to reference material molecular weight logarithm and elution volume, matching obtains regression equation and is: y=-38.628x+195.48 (R
2=0.9958); Wherein, y is elution volume, and x is the logarithmic value of corresponding molecular weight; Get each sample solution, carry out wash-out and detection by the same way, peak volume will be gone out and bring typical curve into, molecular weight analyte and distribution range thereof, by peak area normalization method, determine the percentage contents of different molecular weight protein peptide, acquired results is as shown in table 1.
Table 1: polypeptide molecular weight distributes
| ≥3000Da | 3000～1000Da | 1000～300Da | ＜300Da | |
| Embodiment 1 | -- | 2.8％ | 54.6％ | 42.6 |
| Embodiment 2 | -- | 10.2％ | 49.8％ | 40.0 |
| Embodiment 3 | -- | 5.4％ | 50.9％ | 43.7 |
| Comparative example 1 | 41.6％ | 43.8％ | 9.6％ | 5.0 |
| Comparative example 2 | 32.9％ | 33.7％ | 24.6％ | 9.1 |
| Comparative example 3 | 28.6％ | 26.9％ | 33.4％ | 11.1 |
From above result, woods silicone oil polypeptide molecular weight provided by the invention is little, and homogeneous.
Experimental example 2
1, laboratory sample: by each embodiment and comparative example gained collagen peptide soluble in water, be mixed with the polypeptide solution that concentration is 1%, equal-volume uniform application is in experiment material.
2, experiment material: the Wistar rat of getting body weight 200 ± 20g, anesthesia, picks clean fine hair, careful peeling, is separated subcutaneus adipose tissue and mucous tissue, repeatedly rinses several times with physiological saline; Dispersion device and medium select modified Franz diffusion cells; Dispersive medium selects physiological saline, provides the environment similar in human body.
3, Transdermal absorption experimental implementation conveniently, gets transdermal solution, under the general ultraviolet wavelength 215nm that polypeptide detects, detects the ultraviolet light absorption angle value of solution, is obtained the transmitance of Rana oil polypeptide by formula; Detected result is as shown in table 2.
Table 2: the transmitance of polypeptide
| Sample | Transmitance (%) |
| Embodiment 1 | 90.2 |
| Embodiment 2 | 81.3 |
| Embodiment 3 | 85.4 |
| Comparative example 1 | 37.9 |
| Comparative example 2 | 54.3 |
| Comparative example 3 | 56.2 |
From above result, woods silicone oil polypeptide provided by the invention has good transdermal rate, is suitable for use in skin care product.
Experimental example 3
The oxidation-resistance of polypeptide can be reacted by its elimination efficiency for DPPH.Specifically, DPPH is a kind of stable free radical, and its lone-pair electron have strong absorption near 517nm; When the material with anti-oxidant function exists, lone-pair electron are matched by the reducing substances with anti-oxidant function, and the absorption near 517nm disappears or weakens, by measuring the degree absorbing and weaken, draw free radical scavenging activity, thus the oxidation-resistance of reactive material.
Test method: get each embodiment and be configured to the identical solution of concentration with comparative example gained polypeptide, is that the DPPH solution 2mL of 100 μm of oL/L successively adds in same tool plug test tube by solution 2mL and concentration, shakes up; Left at room temperature 30min, working sample absorbancy under 517nm wavelength.The method of calculation of inhibiting rate are: K%=[1-(Ai-Aj)/Ac] × 100%; Wherein, the absorbancy of Ai:2mLDPPH solution+2mL product to be tested solution, the absorbancy of Aj:2mL product to be tested solution+2mL solvent, the absorbancy of Ac:2mL DPPH solution+2mL solvent.During test, product to be tested being unified weaker concn is 0.1% (w/v).
Known after testing, the free radical scavenging activity of the embodiment of the present invention 1 gained polypeptide is 88.2%, and embodiment 2 and 3 is respectively 81.8% and 83.4%; The free radical scavenging activity of each comparative example is respectively 52.4%, 63.8% and 65.9%, all remarkable in embodiment 1 ~ 3.
Although above with general explanation, embodiment and test, the present invention is described in detail, and on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a wood frog oil antioxidation polypeptide, is characterized in that, described wood frog oil antioxidation polypeptide is prepared from by the method comprised the following steps:
1) get wood frog oil, remove manadesma, add 2 ~ 10 times to the water of wood frog oil volume, be placed in 2 ~ 4 DEG C of bubbles and send out 8 ~ 12h, obtain wood frog oil solution;
2) regulating step 1) pH value of gained wood frog oil solution is 1.6 ~ 1.8, add 0.1 ~ 0.3g/ml stomach en-, at 35 ~ 40 DEG C, abundant enzymolysis, obtains Pepsin enzymolysis solution;
3) regulating step 2) pH value of gained Pepsin enzymolysis solution is 6.0 ~ 7.0, add 0.2 ~ 0.4g/ml neutral protease, at 45 ~ 55 DEG C, abundant enzymolysis, obtains neutral protein enzymolysis liquid;
4) regulating step 3) pH value of gained neutral protease enzymolysis liquid is 7.0 ~ 8.0, add 0.2 ~ 0.4g/ml bromeline, at 55 ~ 60 DEG C, abundant enzymolysis, obtains pineapple enzymolysis solution;
5) by step 4) gained pineapple enzymolysis solution is heated to 70 ~ 75 DEG C, and leave standstill 10 ~ 15min, make enzyme-deactivating, obtain enzymolysis solution;
6) step 5 is got) gained enzymolysis solution, use the membrane filtration of 0.3 ~ 0.4 μm, collect filtrate, re-use the ultrafiltration membrance filter that molecular weight cut-off is 4 ~ 5kD, collect filtrate, after adding gac room temperature stirring at low speed 1 ~ 3h, cross and filter gac, vacuum concentration, spraying dry, obtains wood frog oil antioxidation polypeptide.
2. wood frog oil antioxidation polypeptide according to claim 1, is characterized in that, step 2) described pepsic specific activity is 3000 ~ 5000U/g.
3. wood frog oil antioxidation polypeptide according to claim 2, is characterized in that, step 2) time of described enzymolysis is 4 ~ 5h.
4. wood frog oil antioxidation polypeptide according to claim 1, is characterized in that, step 3) specific activity of described neutral protease is 40000 ~ 70000U/g.
5. wood frog oil antioxidation polypeptide according to claim 4, is characterized in that, step 3) time of described enzymolysis is 3 ~ 4h.
6. wood frog oil antioxidation polypeptide according to claim 1, is characterized in that, step 4) specific activity of described bromeline is 600000 ~ 900000U/g.
7. wood frog oil antioxidation polypeptide according to claim 6, is characterized in that, step 4) time of described enzymolysis is 2 ~ 3h.
8. wood frog oil antioxidation polypeptide according to claim 1, is characterized in that, described wood frog oil antioxidation polypeptide is prepared from by the method comprised the following steps:
1) get wood frog oil, remove manadesma, add 2 ~ 10 times to the water of wood frog oil volume, be placed in 2 ~ 4 DEG C of bubbles and send out 8 ~ 12h, obtain wood frog oil solution;
2) regulating step 1) pH value of gained wood frog oil solution is 1.6 ~ 1.8, the specific activity adding enzyme is 0.1 ~ 0.3g/ml stomach en-of 3000 ~ 5000U/g, and enzymolysis 4 ~ 5h at 35 ~ 40 DEG C, obtains Pepsin enzymolysis solution;
3) regulating step 2) pH value of gained Pepsin enzymolysis solution is 6.0 ~ 7.0, the specific activity adding enzyme is 0.2 ~ 0.4g/ml neutral protease of 40000 ~ 70000U/g, and enzymolysis 3 ~ 4h at 45 ~ 55 DEG C obtains neutral protein enzymolysis liquid;
4) regulating step 3) pH value of gained neutral protease enzymolysis liquid is 7.0 ~ 8.0, the specific activity adding enzyme is 0.2 ~ 0.4g/ml bromeline of 600000 ~ 900000U/g, and enzymolysis 2 ~ 3h at 55 ~ 60 DEG C, obtains pineapple enzymolysis solution;
5) by step 4) gained pineapple enzymolysis solution is heated to 70 ~ 75 DEG C, and leave standstill 10 ~ 15min, make enzyme-deactivating, obtain enzymolysis solution;
6) step 5 is got) gained enzymolysis solution, use the membrane filtration of 0.3 ~ 0.4 μm, collect filtrate, re-use the ultrafiltration membrance filter that molecular weight cut-off is 4 ~ 5kD, collect filtrate, after adding gac room temperature stirring at low speed 1 ~ 3h, cross and filter gac, vacuum concentration, spraying dry, obtains wood frog oil antioxidation polypeptide.
9. wood frog oil antioxidation polypeptide according to claim 1, is characterized in that, described wood frog oil antioxidation polypeptide is prepared from by the method comprised the following steps:
1) get wood frog oil, remove manadesma, add 8 times to the water of wood frog oil volume, be placed in 3 DEG C of bubbles and send out 10h, obtain wood frog oil solution;
2) regulating step 1) pH value of gained wood frog oil solution is 1.7, the specific activity adding enzyme is the 0.2g/ml stomach en-of 4000U/g, and enzymolysis 4.5h at 38 DEG C, obtains Pepsin enzymolysis solution;
3) regulating step 2) pH value of gained Pepsin enzymolysis solution is 6.5, the specific activity adding enzyme is the 0.3g/ml neutral protease of 60000U/g, and enzymolysis 3.5h at 50 DEG C obtains neutral protein enzymolysis liquid;
4) regulating step 3) pH value of gained neutral protease enzymolysis liquid is 7.5, the specific activity adding enzyme is the 0.3g/ml bromeline of 800000U/g, and enzymolysis 2.5h at 58 DEG C, obtains pineapple enzymolysis solution;
5) by step 4) gained pineapple enzymolysis solution is heated to 72 DEG C, and leave standstill 12min, make enzyme-deactivating, obtain enzymolysis solution;
6) step 5 is got) gained enzymolysis solution, use the membrane filtration of 0.35 μm, collect filtrate, re-use the ultrafiltration membrance filter that molecular weight cut-off is 4kD, collect filtrate, after adding gac room temperature stirring at low speed 2h, cross and filter gac, vacuum concentration, spraying dry, obtains wood frog oil antioxidation polypeptide.
10. wood frog oil antioxidation polypeptide described in claim 1 ~ 9 any one is preparing the application in makeup, food, healthcare products or medicine.
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| CN105420331A (en) * | 2016-01-15 | 2016-03-23 | 北京博肽未名生物技术有限公司 | Method for extracting polypeptide through tadpole enzymolysis and polypeptide prepared by method |
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| CN107136516A (en) * | 2017-04-20 | 2017-09-08 | 国药（延边）林蛙生物科技有限公司 | A kind of preparation method of oviductus ranae peptide particulate |
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| CN107308436B (en) * | 2017-05-23 | 2021-10-26 | 辽宁阿里郎生物工程股份有限公司 | Biological product with physical fatigue relieving and antioxidant functions and preparation method thereof |
| CN115737516A (en) * | 2022-11-25 | 2023-03-07 | 哈尔滨欧替药业有限公司 | Anti-aging whitening moisturizing composition and essence emulsion containing oviductus ranae hydrolysate and preparation method thereof |
| CN115778853A (en) * | 2022-12-07 | 2023-03-14 | 哈尔滨欧替药业有限公司 | Anti-aging essence containing oviductus ranae enzymolysis peptide and preparation method thereof |
| CN116855309A (en) * | 2023-08-04 | 2023-10-10 | 通化康元生物科技有限公司 | Instant oviductus ranae and preparation method thereof |
| CN116855309B (en) * | 2023-08-04 | 2023-12-19 | 通化康元生物科技有限公司 | Instant oviductus ranae and preparation method thereof |
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