CN112266943B - Extraction method and application of polypeptide from wild goose palmate yellow skin - Google Patents
Extraction method and application of polypeptide from wild goose palmate yellow skin Download PDFInfo
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- CN112266943B CN112266943B CN202011167186.1A CN202011167186A CN112266943B CN 112266943 B CN112266943 B CN 112266943B CN 202011167186 A CN202011167186 A CN 202011167186A CN 112266943 B CN112266943 B CN 112266943B
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Abstract
An extraction method of polypeptide from sea-goose palmate wampee and application thereof belong to the technical field of animal polypeptide extraction and development, and in particular relate to the extraction of polypeptide from sea-goose palmate wampee and application thereof in preparing antifreezing skin cream. The polypeptide extraction process comprises the following steps: degreasing with petroleum ether, hydrolyzing to obtain the protein of the palmtop yellow skin of the sea geese, and carrying out equilibrium dialysis and purification to obtain the polypeptide extract of the palmtop yellow skin of the Xiang Haiyan. The invention has the advantages of simple manufacturing flow and low cost, and the cold injury resistant activity of the polypeptide to the sea goose palmate cortex is proved to be a certain cold injury resistant activity in an experiment of examining the cold injury resistant activity of the polypeptide by using an in vitro cold injury cell model. The cold injury-resistant skin cream can be applied to the skin cream for resisting cold injury, and can reduce the occurrence rate of cold injury. The discarded palms of the sea geese are utilized, so that the waste of biological resources and environmental pollution are reduced.
Description
Technical Field
The invention belongs to the technical field of animal polypeptide extraction and development, and particularly relates to an extraction method of polypeptide from wild goose palmate wampee and application thereof.
Background
Sea-going geese (Anser cygnoides) is a species belonging to the genus Anser cygnoideae, class Anseris, which is a species specific to Jilin province, and which has been identified as a new species by the national center for biotechnology information in 2015 (Genbank ID: 1856198) as living in the natural protection area of sea-going elm county of Jilin province throughout the year. The wild goose has the unique characteristic that the bird waiting does not migrate with seasons under artificial domestication, and can resist severe cold at-40 ℃ in winter.
The yellow skin of Yan palmar is a yellow skin on the foot sole and the foot web of Yan palmar, and is recorded in the ancient books of China such as Ben Cao gang mu and Qian jin Ding Zhi. The literary composition recorded in Ben Cao gang mu is: the toe joint is baked and ground and then wet and rotten. Baking, oil mixing and Tu Dongchuang. "
At present, the research reports on the sea-geese at home and abroad are very rare, and the sea-geese have certain research value on feathers, meat, blood, bones and fat of the sea-geese, but are mostly used as raw material outlets, and the sea-geese palmtop yellow skin is discarded as waste. The Jilin province white city is used as the biggest breeding base of the sea-facing geese in China, and the number of the sea-facing geese killed each year is about 30 ten thousand. The discarding of the palmtop yellow skin of the sea geese results in great waste of biological resources and environmental pollution.
There is a need in the art for a new solution to this problem.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: provides a method for extracting polypeptide from the palmtop yellow skin of the sea-geese and application thereof, which are used for solving the technical problems of great waste of biological resources and environmental pollution caused by discarding the palmtop yellow skin of the sea-geese.
A method for extracting polypeptide from palmar yellow skin of sea goose comprises the following steps sequentially,
step one, selecting optimal protease;
hydrolyzing the defatted and dried palmtop yellow skin of the wild goose for 5 hours by using the selected optimal protease according to a set feed liquid ratio, and obtaining a hydrolysate of the palmtop Huang Pidan of the wild goose by using the hydrolyzed liquid;
step three, placing the yellow skin protein hydrolysate of the wild goose palms obtained in the step two into a dialysis bag for balanced dialysis for 48 hours, wherein the obtained liquid outside the dialysis bag is the hydrolysate of the wild goose palms Huang Pidan;
and step four, utilizing gel exclusion chromatography to determine that the wild goose palmar Huang Pidan hydrolysate obtained in the step three is the wild goose palmar polypeptide extract, and freeze-drying the wild goose palmar polypeptide extract obtained in the step three for later use.
The specific method for selecting the optimal protease is that the acidic protease, the neutral protease and the alkaline protease with the same quality are weighed, the equal amount of the goose palmetto skin is hydrolyzed under the same condition, and the degree of hydrolysis is measured, wherein the value of the neutral protease is the highest, and the neutral protease is the optimal protease.
The acid protease comprises pepsin.
The specific degreasing and drying method for the palmtop yellow skin of the sea geese in the second step comprises the following steps: the yellow skin of the oriented sea-geese is completely immersed in petroleum ether with the boiling range of 60-90 ℃ for degreasing for 48 hours, and is dried to constant weight at 60 ℃ after being washed to be neutral by purified water, so that the yellow skin of the oriented sea-geese after degreasing and drying is obtained.
And in the second step, the set feed liquid ratio is 1:15.
The application of polypeptide to the palmar yellow skin of the sea geese is applied to the skin cream for resisting cold injury.
Through the design scheme, the invention has the following beneficial effects:
the extraction method disclosed by the invention utilizes the wild goose palmate wampee to obtain the Xiang Haiyan wild goose palmate wampee polypeptide, and has the advantages of simple manufacturing flow and low cost. The wild goose palmate wampee polypeptide has certain activity in the aspect of cold injury resistance application, and the wild goose palmate wampee polypeptide extract prepared by the method is applied to cold injury resistance skin cream, so that the occurrence rate of cold injury can be reduced. The discarded palms of the sea geese are utilized, so that the waste of biological resources and environmental pollution are reduced.
Drawings
The invention is further described with reference to the drawings and detailed description which follow:
fig. 1 is a graph showing the effect of the polypeptide on LDH on the polypeptide of the palms of the sea-geese and the palms of the sea-geese in a second embodiment of the invention.
Fig. 2 is a graph showing the influence of the polypeptide on the NO content of the polypeptide of the palms of the sea-geese and the palms of the sea-geese in a second embodiment of the method for extracting the polypeptide of the palms of the sea-geese and the application of the method.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Embodiment one: extracting polypeptide from the palmate yellow skin of the sea goose.
Step 1, weighing different proteases with the same quality, hydrolyzing the palmtop yellow skin of the sea geese under the same condition, and screening out the most suitable protease;
the specific method for screening the optimal protease comprises the steps of weighing pepsin, neutral protease and alkaline protease with the same quality, hydrolyzing the equal amount of the yellow skin of the wild goose palmetto under the same condition, and measuring the degree of hydrolysis, wherein the value of the neutral protease is highest, and the neutral protease is the optimal protease.
Step 2, according to 1:15, degreasing petroleum ether (boiling range is 60-90 ℃) for 48 hours by using neutral protease screened in the step 1, drying to constant weight, hydrolyzing the yellow skin of the sea-geese after degreasing and drying, and collecting hydrolysate.
And step 3, balancing and dialyzing the hydrolysate obtained in the step 2 in a dialysis bag 48, and taking the dialyzing external liquid.
And 4, feeding the dialyzed and purified outer liquid in the step 3 into gel exclusion chromatography, determining the molecular weight of the extract in the step 3 to obtain the polypeptide extract of the wild goose palmate skin, and freeze-drying for later use.
Embodiment two: and testing cold injury resistance activity of the polypeptide of the palmate yellow skin of the sea geese.
The cold injury resistance activity test is carried out by adopting the wild goose palmate yellow skin protein and the wild goose palmate yellow skin polypeptide respectively.
The rat vascular endothelial cells in logarithmic phase are cooled to 0 ℃, -5 ℃, -10 ℃, -15 ℃, -20 ℃ for 5min respectively by taking 4 ℃ as an initial temperature, and are rewarmed for 10min at 37 ℃ for continuous culture. Taking the non-cooled group as a control group, adding 20 mu L of MTT solution cell culture boxes into each hole of the cold injury model group at 0 ℃,5 ℃, 10 ℃, 15 ℃ and 20 ℃ respectively, continuously culturing for 4 hours, and sucking the supernatant. To each well, 150. Mu.L of dimethyl sulfoxide (DMSO) was added, and the mixture was shaken at a low speed for 300s and allowed to stand for a while. Cell viability was calculated. Cell culture supernatants from each group were collected to determine serum lactate dehydrogenase LDH activity.
Preparing polypeptide solutions of the polypeptide of the palms of the sea-wild geese and the yellow skin with the concentration of 50 mug/mL, 100 mug/mL, 200 mug/mL and 400 mug/mL respectively, adding the solutions into an established rat vascular endothelial cell cold injury model, and adding 5% CO at 37 DEG C 2 Cell culture supernatant was taken after continuous culture in the incubator for 24 hours to determine LDH activity and nitric oxide NO content.
Detection of Activity criteria: the LDH activity is reduced, and the increase of the NO content indicates that the polypeptide has cold injury resistance to the polypeptide of the palmate yellow skin of the sea geese.
The cold injury resistance test result of the polypeptide of the palmate yellow skin of the sea goose is shown in fig. 1 and 2. The experimental results shown in fig. 1 and fig. 2 show that the polypeptide has a certain cold injury resistance activity to the polypeptide of the palmetto cortex of the sea goose, can reduce the LDH activity in a dose-dependent manner and increase the NO content. Therefore, the polypeptide of the palmar yellow skin of the wild goose can be applied to the skin cream for resisting cold injury.
The above examples represent only a few embodiments of the present invention, which are described in more detail and in particular, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the claims appended hereto.
Claims (3)
1. A method for extracting polypeptide from wild goose palmate cortex, which is characterized by comprising the following steps: comprising the following steps, and the following steps are carried out in sequence,
step one, selecting the most suitable protease
Weighing acid protease, neutral protease and alkaline protease with the same mass, hydrolyzing the equal amount of the yellow skin of the wild goose palmetto to the sea goose under the same condition, and measuring the degree of hydrolysis, wherein the value of the neutral protease is highest, and the neutral protease is the most suitable protease;
step two, fully immersing the oriented sea-goose palmar yellow skin in petroleum ether with the boiling range of 60-90 ℃ for degreasing for 48 hours, washing the sea-goose palmar yellow skin to be neutral by purified water, and drying the sea-goose palmar yellow skin at the temperature of 60 ℃ to obtain degreased and dried sea-goose palmar yellow skin, hydrolyzing the degreased and dried sea-goose palmar yellow skin with selected optimal protease for 5 hours according to a set feed liquid ratio, and obtaining a sea-goose palmar Huang Pidan hydrolysate from the hydrolysate, wherein the set feed liquid ratio is 1:15;
step three, placing the yellow skin protein hydrolysate of the wild goose palms obtained in the step two into a dialysis bag for balanced dialysis for 48 hours, wherein the obtained liquid outside the dialysis bag is the hydrolysate of the wild goose palms Huang Pidan;
and step four, performing gel exclusion chromatography on the yellow skin protein hydrolysate of the wild goose palms Huang Pidan of the wild geese, which is obtained in the step three, to determine that the yellow skin polypeptide extract of the wild geese is obtained by the white skin polypeptide extract of the wild geese through molecular weight, and freeze-drying the yellow skin polypeptide extract of the wild geese for later use.
2. The method for extracting polypeptide from palmtop yellow skin of sea geese as claimed in claim 1, wherein the method comprises the following steps: the acid protease comprises pepsin.
3. An application of polypeptide extracted by the method of claim 1 to the sea goose palmate cortex phellodendri, which is characterized in that: is applied to preparing the skin cream for resisting cold injury.
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| CN106544385B (en) * | 2015-09-16 | 2020-11-27 | 上海理工大学 | Method for separating collagen antifreeze peptide |
| CN109251955A (en) * | 2017-07-13 | 2019-01-22 | 青岛万图明生物制品有限公司 | The preparation process of birds collagen peptide |
| CN107375173A (en) * | 2017-07-29 | 2017-11-24 | 北京健旭康技术有限公司 | Purposes of the special cultivation animal in cosmetics are prepared |
| CN109504730A (en) * | 2018-12-14 | 2019-03-22 | 金华市艾力生物科技有限公司 | A kind of extracting method of fish-skin active peptides |
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Non-Patent Citations (2)
| Title |
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| Antifreeze activity of collagens from different sources;Cong Thanh Nguyen等;Food Science;第35卷(第17期);22-26 * |
| Antifreeze and cryoprotective activities of ice-binding collagen peptides from pig skin;Hui Cao等;FOOD CHEMISTRY;第1卷(第1期);1245-1253 * |
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