CN111903985A

CN111903985A - Preparation method and application of protein oligopeptide rich in desmosine and isodesmosine

Info

Publication number: CN111903985A
Application number: CN202010800475.4A
Authority: CN
Inventors: 杨红菊
Original assignee: Anhui Shengmeinuo Biology Technology Co ltd
Current assignee: Anhui Shengmeinuo Biology Technology Co ltd
Priority date: 2020-08-11
Filing date: 2020-08-11
Publication date: 2020-11-10

Abstract

The invention relates to the technical field of polypeptide extraction, in particular to a preparation method and application of protein oligopeptide rich in desmosine and isodesmosine. The invention provides a preparation method of protein oligopeptide, which comprises the steps of sequentially soaking tissues to be extracted in alkali liquor and acid liquor, grinding the tissues to the granularity of 100-200 meshes, carrying out enzymolysis by using alkaline protease and pancreatin, carrying out enzyme deactivation and centrifugation to obtain supernatant, and carrying out fine filtration and nanofiltration to obtain a solution containing the protein oligopeptide; wherein the tissue to be extracted is an arterial duct, ligament, lung, fascia, cartilage or eggshell membrane. The extracted protein oligopeptide is rich in desmosine and isodesmosine, and the polypeptide with the molecular weight less than 1000Da accounts for 50-85% of the total polypeptide mass. The method uses alkali solution and acid solution for degreasing and removing foreign protein, avoids residue and pollution of organic solvent, and has simple operation and high extraction efficiency.

Description

Preparation method and application of protein oligopeptide rich in desmosine and isodesmosine

Technical Field

The invention relates to the technical field of polypeptide extraction, in particular to a preparation method and application of protein oligopeptide rich in desmosine and isodesmosine.

Background

The skin is the largest organ of the human body, covers the whole body, has functions of absorption, secretion, excretion, sensation, participation in metabolism and the like, and is an important barrier and defense organ. The skin aging seriously affects the health and beauty of people and is a hot problem of general attention and research of people. Among them, skin photoaging is a change in structure and function caused by repeated exposure of the skin to ultraviolet rays for a long period of time. Skin aging is manifested clinically mainly by rough skin, sagging, drooping, and even the appearance of tumors. The pathological manifestations are uneven epidermal layer thickness, flat epidermal dermal interface, telangiectasia, collagen fiber breakage, disorder, accelerated ultraviolet-induced collagen decomposition, and continuous decrease of collagen content of type I and type III (ColI and ColIII). similar to collagen, elastin is lost with age, and ultraviolet irradiation also causes elastic fiber winding and fragmentation. Unlike natural skin aging, photoaging can be ameliorated and reversed by means of drugs or sunscreens.

Elastin, a structural protein, is the most stable protein in the extracellular matrix and is present in a number of connective tissues, accounting for 2-4% of the total skin protein. The precursor of elastin, tropoelastin, is a soluble monomer with 60-70 kDa, and the elastin is an insoluble polymer formed by crosslinking Desmosine (DES) and Isodesmosine (IDS) formed by oxidizing 2-4 tropoelastin through four lysine residues, and has stable structure. Elastin and related proteins constitute elastic microfibrils, which in turn constitute elastic fibers with amorphous elastin. The elastic fibers and the collagen fibers are tightly connected and mutually interwoven, and the collagen gives the skin strength and stretch resistance; the special cross-linking and hydrophobic structure of elastin endows the elastin with good elasticity and extensibility, and enables organs such as skin and the like to maintain flexible elasticity.

The elastin has stable structure and is insoluble in water, weak acid, weak base, SDS and other conventional protein solvents. Therefore, the isolation of elastin by complicated and harsh conditions is currently studied because it is difficult to characterize elastin by molecular weight, isoelectric point, etc. because of insolubility, and its purity can only be evaluated by amino acid composition. Elastin is rich in nonpolar amino acids such as glycine, alanine, proline, valine, etc., and unique cross-linked amino acids DES and IDS. However, it contains only a very small amount of hydroxyproline (Hyp) compared to collagen (about 1%).

Animal tissues rich in elastin, including arterial vessels, ligaments, tendons, etc., have long been consumed as food. But has high fat content, poor digestion and absorption of human body and low bioavailability. The elastin peptide prepared by the enzymolysis process reported at present is proved to have the function of improving the skin and blood vessel state through experiments, but the processes either use volatile organic solvents or use non-food enzymes which are difficult to convert. And the elastin is not separated from the collagen, glycoprotein, globulin and the like, and before the stable elastin is hydrolyzed, the collagen, glycoprotein, globulin and the like which coexist with the stable elastin are deeply hydrolyzed into oligopeptide, free amino acid and even generate small-molecule nitrogenous substances, so that the product quality is influenced.

The invention tries to hydrolyze elastin in edible animal tissues into small molecular polypeptides, so as to enhance the solubility of the small molecular polypeptides, make the small molecular polypeptides become high-purity protein peptide powder which is convenient to absorb, and improve the bioavailability of the small molecular polypeptides. And discusses the effects of improving skin photoaging and promoting collagen secretion.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a preparation method and application of protein oligopeptide rich in desmosine and isodesmosine.

The invention provides a protein oligopeptide rich in desmosine and isodesmosine, which comprises polypeptide, free amino acid and ash;

in the polypeptide and the free amino acid, the sum of the mass fractions of desmosine and isodesmosine is 0.3-0.8%;

in the polypeptides, the mass fraction of the polypeptides with the molecular weight less than 1000Da is 50-85%, the mass fraction of the polypeptides with the molecular weight of 1000 Da-3000 Da is 8-25%, and the mass fraction of the polypeptides with the molecular weight more than 3000Da is 2-15%.

In the protein oligopeptide provided by the invention, the content of elastin characteristic amino acid desmosine and isodesmosine is high, while the content of collagen characteristic amino acid hydroxyproline is low, the mass fraction is lower than 2%, and is far lower than the content (about 10%) of collagen, which indicates that the elastin purity in the protein oligopeptide provided by the invention is high.

The preparation method of the protein oligopeptide comprises the following steps:

sequentially soaking the tissue to be extracted in alkali liquor and acid liquor, grinding to 100-200 meshes, carrying out enzymolysis by using alkaline protease and pancreatin, carrying out enzyme deactivation and centrifugation to obtain supernatant, and filtering and nano-filtering to obtain a solution containing protein oligopeptide; wherein the tissue to be extracted is an arterial duct, ligament, cartilage, lung, fascia or eggshell membrane.

In the preparation method provided by the invention, the tissue to be extracted is from pigs, cattle, sheep, chickens, ducks, geese or fishes.

In the preparation method provided by the invention, the degreasing adopts a method of washing with alkali liquor and acid liquor, so that the problems of environmental pollution caused by organic solvent and organic solvent residue in the product are avoided.

In the preparation method of the invention, the alkali liquor is NaHCO₃Solution of NaHCO₃The mass fraction of the alkali liquor is 0.5 wt%, and the alkali liquor is soaked for 0.5 to 2 hours at normal temperature;

the acid solution is citric acid or hydrochloric acid, and the acid solution is soaked under the conditions that the pH value is 2.2-3 and the temperature is 75-85 ℃ for 1-3 hours.

The tissue to be extracted is crushed to a particle size of 0.5cm before extraction. The mass-volume ratio of the tissue to be extracted to the alkali liquor is (2-3) L/kg. After being soaked in alkali liquor, the mixture is filtered and washed, and then is soaked in acid liquor. The volume of the acid solution is 1-1.5 times of that of the alkali solution.

The invention utilizes the characteristics of acid resistance and heat resistance of the elastin in a certain range and the changeability of proteins such as glycoprotein, globulin and collagen under acid and heat conditions, removes other proteins such as glycoprotein, globulin and collagen to the maximum extent by selecting the pH value and the temperature in a proper range and the type, the proportion and the dosage of the protease, purifies the elastin, ensures that the protease is easier to contact with the elastin in the oligopeptide preparation process, shortens the enzymolysis time, effectively reduces free amino acids and low molecular weight nitrogenous substances generated by deep hydrolysis of the protein, improves the product purity and improves the product quality. The high-purity protein oligopeptide rich in DES and IDS obtained by enzymolysis has small molecules and is easier to be absorbed by human bodies.

In the preparation method, the mass ratio of the alkaline protease to the pancreatin is 4: 1; the enzymolysis condition is that the pH is 7.8-8.5; enzymolysis is carried out for 5-6 h at 55-60 ℃.

In some embodiments, the concentration of the alkaline protease is 0.8-1 g/L. The concentration of the pancreatin is 0.2-0.25 g/L.

After further fine filtration and nanofiltration desalination, the product is not easy to absorb moisture, and the product quality is improved.

In the preparation method, the centrifugation condition is 4500r/min centrifugation for 20 min; the fine filtration process is that the centrifugal supernatant is filtered by filter membranes with the diameter of 10 mu m and 0.45 mu m in turn; the nanofiltration has a molecular weight cutoff of 160Da, a pressure of 0.5MPa and a time of 5 min.

In the preparation method, after the grinding to the granularity of 100-200 meshes and before the enzymolysis by using alkaline protease and pancreatin, the method also comprises the step of digesting the collagen by enzymolysis; the enzyme for dissolving out the collagen is papain or a special enzyme for hydrolyzing the collagen. The concentration of the papain is 0.1-0.3 wt%, and the enzymolysis conditions are that the pH value is 6.8, the temperature is 45 ℃, and the time is 2 hours. The enzymolysis condition of the special enzyme for collagen hydrolysis is that the pH value is 7.2, the temperature is 60 ℃, and the time is 2 hours.

In the preparation method, the solution containing the protein oligopeptide is subjected to reduced pressure concentration until the solid content is 35-50%, and then is subjected to spray drying to obtain the protein oligopeptide; the inlet temperature of the spray drying is 180 ℃ and the outlet temperature is 70 ℃.

Experiments prove that the protein oligopeptide rich in DES and IDS can obviously prevent and improve skin photoaging caused by ultraviolet radiation.

The invention also provides the application of the protein oligopeptide or the protein oligopeptide prepared by the preparation method in preparing food, medicine and/or cosmetics.

The invention also provides food, medicine and/or cosmetics containing the protein oligopeptide or the protein oligopeptide prepared by the preparation method.

The invention provides a preparation method of protein oligopeptide, which comprises the steps of sequentially soaking tissues to be extracted in alkali liquor and acid liquor, grinding the tissues to the granularity of 100-200 meshes, carrying out enzymolysis by using alkaline protease and pancreatin, carrying out enzyme deactivation and centrifugation to obtain supernatant, and carrying out fine filtration and nanofiltration to obtain a solution containing the protein oligopeptide; wherein the tissue to be extracted is artery duct, ligament, fascia, lung, cartilage and eggshell membrane. The extracted protein oligopeptide is rich in desmosine and isodesmosine, and the polypeptide with the molecular weight less than 1000Da accounts for 50-85% of the total polypeptide mass. The method uses alkali solution and acid solution for degreasing, avoids residue and pollution of organic solvent, and has simple operation and high extraction efficiency.

Detailed Description

The invention provides a preparation method and application of a protein oligopeptide rich in desmosine and isodesmosine, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the preparation method. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The method for preparing the protein oligopeptide rich in DES and IDS comprises the following steps:

(1) pretreatment: cleaning main artery and ligament of cattle/pig with removed attached muscle, crushing into small pieces of about 0.5cm, and adding 0.5% NaHCO 2-3 times of the weight of the crushed meat₃Stirring for 0.5-2 h, removingRemoving impurities such as fat, soluble protein and the like, filtering and cleaning;

(2) and (3) elastin purification: adding water into the cleaned minced meat, adjusting the pH value to acidity, preserving the temperature for 2 hours, filtering and washing; adding protease to hydrolyze un-dissolved collagen if necessary, filtering and washing with water. Adding water into the residual powder, adjusting the pH value to be neutral, and grinding the mixture to be 100-200 meshes by using a colloid mill;

(3) protein peptide preparation: adjusting the pH value of the slurry, adding compound protein for enzymolysis, and inactivating enzyme; centrifuging; fine filtering, and desalting the filtrate by nanofiltration; concentrating under reduced pressure; and (5) spray drying.

Preferably, the addition amount of water in the step (2) is 2-5 times of the volume of the minced meat, the acidic pH value range is 2-4, and the heat preservation temperature is 70-85 ℃.

Preferably, the protease selected in the step (2) is papain or a collagen hydrolysis dedicated enzyme, the addition amount of the enzyme is preferably 0.1-0.3%, the pH value is 6.8-8.0, and the temperature is 45-60 ℃.

The protease used in step (3) is a combination of an alkaline protease and pancreatin, preferably a combination of an alkaline protease and pancreatin in a ratio of 4: 1. The addition amount of the combined protease is 0.1-0.5% of the mass of the raw materials. The preferable enzymolysis condition is that the pH value is 7.5-9.0, the temperature is 40-60 ℃, and the time is 4-6 h.

In the step (3), the filtrate is continuously subjected to constant volume percolation through a nanofiltration device with the molecular weight cutoff of 160Da, the operation pressure is 0.4-0.8 MPa, preferably 0.5MPa, and the time is 5 min.

The elastin oligopeptide obtained by the method has the protein content of more than or equal to 90 percent, the ash content of less than 4 percent and is rich in DES and IDS, the sum of the contents of the DES and the IDS is between 0.3 and 0.8 percent, the content of hydroxyproline (Hyp) is less than 2 percent and is far lower than the Hyp content (about 10 percent) of collagen, and the elastin has high purity.

The elastin oligopeptide rich in DES and IDS obtained by the method has the molecular weight of 50-85% below 1000Da, the molecular weight of 8-25% between 1000Da and 3000Da and the molecular weight of 2-15% above 3000 Da. Can improve the moisture of skin oil of mice irradiated by ultraviolet, partially maintain the skin elasticity and the content of collagen fibers, improve the ratio of ColI/ColIII, and effectively prevent and relieve the skin photoaging of the mice.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

Wherein, the special enzyme for collagen hydrolysis is from Nanning Dong Henghuadao biotechnology, Inc.

The invention is further illustrated by the following examples:

example 1 DES and IDS enriched bovine arterial protein oligopeptide

Taking 2.03kg of bovine aorta tube without muscle, crushing into small pieces of about 0.5cm, adding 4L of 0.5% NaHCO₃The solution is stirred and soaked for 1.5h, filtered and washed with water. Adding 5L of water, adjusting the pH value to 2.5, preserving the heat at 78 ℃ for 2h, filtering and washing with water. Adding 4L of water into the filter residue, adjusting pH to 6.8, adding 0.1% papain, keeping the temperature at 45 ℃ for 2h, filtering and washing with water. 6L of water was added to the remaining powder, and the mixture was colloid-milled to 100 mesh. The pH of the slurry was adjusted to 8.0, 6g of alkaline protease and 1.5g of pancreatin were added, and the mixture was subjected to enzymatic hydrolysis at 55 ℃ and pH7.8 for 6 hours. After the heat preservation is finished, heating the enzymolysis liquid to 85 ℃, maintaining for 30min for enzyme deactivation, centrifuging at 4500r/min for 20min, and filtering the supernatant with 10 mu m and 0.45 mu m filter membranes. The obtained filtrate is continuously subjected to constant volume and nanofiltration equipment with the molecular weight cutoff of 160Da, the operation pressure is 0.5MPa, and the time is 5 min. And concentrating the nanofiltration trapped fluid under reduced pressure until the solid content is 45 percent. Spray drying at inlet temperature of 180 deg.C and outlet temperature of 70 deg.C.

227g of DES and IDS-rich protein oligopeptide was obtained by the above steps. The elastin oligopeptide has a protein content of 97.2% (dry basis) and an ash content of 1.7%. The proportion of the molecular weight is more than 3000Da is 6.87%, the proportion of 3000 Da-1000 Da is 16.2%, the proportion of 1000 Da-180 Da is 73.1%, the proportion of less than 180Da (free amino acid and the like) is 3.83%, and the average molecular weight is 900 Dalton. The content of DES and IDS in the oligopeptide is 0.42%, and the content of hydroxyproline is 1.27%.

Comparative example bovine arterial elastin peptide without elastin purification

Taking 2.5kg of cow aorta tube without muscle, crushing into small pieces of about 0.5cm, adding 7L of 0.5% NaHCO₃The solution is stirred and soaked for 1h, filtered and washed with water. Adding 12L of waterAging at 95 deg.C for 10min, and grinding with colloid mill until the temperature is reduced to below 50 deg.C to completely pass through 100 mesh. Adjusting pH of the slurry to 7.9, adding 10g alkaline protease and 2.5g pancreatin, and performing enzymolysis at 60 deg.C and pH of 7.9 for 6 h. Heating the enzymolysis solution to 85 deg.C, maintaining for 30min, and inactivating enzyme. Cooling, centrifuging at 4500r/min for 20min, and fine filtering the supernatant with 10 μm and 0.45 μm filter membrane. Continuously percolating the filtrate with constant volume through nanofiltration equipment with molecular weight cutoff of 160Da, operating pressure of 0.6MPa, and time of 6 min. The trapped solution was concentrated under reduced pressure to a solids content of 41%. Spray drying at inlet temperature of 180 deg.C and outlet temperature of 70 deg.C.

283g of protein peptide powder without elastin purification is obtained by the steps. The elastin peptide, protein content 96.6% (on a dry basis), ash content 1.5%. Average molecular weight 923kDa, the proportion of molecular weight > 3000Da is 2.49%, the proportion of 3000 Da-1000 Da is 15.7%, the proportion of 1000 Da-180 Da is 74.8%, the proportion of < 180Da (free amino acids and the like) is 7.07%, the DES and IDS content is 0.26%, and the hydroxyproline content is 4.73%.

Example 2 DES and IDS enriched bovine ligament protein oligopeptides

Collecting 3.5kg of cattle neck ligament without muscle, crushing into small pieces of about 0.5cm, adding 7L of 0.5% NaHCO₃The solution is stirred, soaked for 0.5h, filtered and washed with water. Adding 10L of water, adjusting the pH value to 3.0, keeping the temperature at 85 ℃ for 2h, filtering and washing with water. 15L of water was added to the remaining powder, the pH was adjusted to neutral, and the mixture was ground to 100 mesh with a colloid mill. The pH of the slurry was further adjusted to 8.3, 12g of alkaline protease and 3g of pancreatin were added, and the temperature was maintained at 60 ℃ and pH8.0 for 5 hours. After the heat preservation is finished, heating the enzymolysis liquid to 95 ℃ and maintaining for 15min to inactivate enzyme, centrifuging at 4500r/min for 20min, and filtering the supernatant with 10 mu m and 0.45 mu m filter membranes. The permeate liquid is continuously percolated with constant volume through a nanofiltration device with the molecular weight cutoff of 160Da, the operation pressure is 0.6MPa, and the time is 5 min. And concentrating the nanofiltration trapped fluid under reduced pressure until the solid content is 43 percent. Spray drying at inlet temperature of 180 deg.C and outlet temperature of 70 deg.C.

936g of DES and IDS-rich bovine ligamentin oligopeptide powder was obtained by the above process. The elastin oligopeptide has a protein content of 95.8% (dry basis) and an ash content of 2.1%. Average molecular weight of 950Dalton, wherein the proportion of molecular weight > 3000Da is 5.62%, that of 3000 Da-1000 Da is 19.9%, that of 1000 Da-180 Da is 71.9%, and that of < 180Da (free amino acid, etc.) is 2.62%. DES and IDS content was 0.66%, hydroxyproline content 1.19%.

Example 3 DES and IDS enriched porcine arterial protein oligopeptides

Collecting 3.2kg of pig artery without muscle, crushing into small pieces of about 0.5cm, adding 7L of 0.5% NaHCO₃The solution is stirred and soaked for 2 hours, filtered and washed with water. Adding 7L of water, adjusting the pH value to 2.2, preserving the heat at 85 ℃ for 2h, filtering and washing with water. Adding 7L of water into the minced meat, adjusting pH to 7.2, adding 6.5g of special enzyme for collagen hydrolysis, keeping the temperature at 60 ℃ for 2h, filtering and washing with water. The retentate was added with 12L of water and colloid milled to 100 mesh. The pH of the slurry was adjusted to 8.7, 10g of alkaline protease and 2.5g of pancreatin were added, and the mixture was incubated at 60 ℃ and pH8.5 for 5 hours. Heating the enzymolysis solution to 90 deg.C after heat preservation, maintaining for 15min for inactivating enzyme, centrifuging at 4500r/min for 20min, and filtering the supernatant with 10 μm and 0.45 μm filter membrane. The permeate liquid is continuously percolated with constant volume through a nanofiltration device with the molecular weight cutoff of 160Da, the operation pressure is 0.6MPa, and the time is 5 min. And concentrating the nanofiltration trapped fluid under reduced pressure until the solid content is 43 percent. Spray drying at inlet temperature of 180 deg.C and outlet temperature of 70 deg.C.

366g of DES and IDS enriched aortic protein oligopeptides were obtained by the above procedure. The elastin oligopeptide powder has a protein content of 95.4% (dry basis) and an ash content of 2.1%. Average molecular weight of 972Dalton, wherein the proportion of molecular weight > 3000Da is 4.33%, that of 3000 Da-1000 Da is 16.2%, that of 1000 Da-180 Da is 72.6%, and that of < 180Da (free amino acid, etc.) is 3.79%. DES and IDS content was 0.43%, hydroxyproline content 1.31%.

Efficacy verification

1. Research for improving mouse skin photoaging by protein oligopeptide rich in DES and IDS

60 adult female mice were randomly divided into 6 groups, respectively: control group, model + protein oligopeptide group obtained in example 1, model + protein oligopeptide group obtained in comparative example, model + protein oligopeptide group obtained in example 2, and model + protein oligopeptide group obtained in example 3. Starting purple after 1 week of acclimatizing feedingExternal irradiation modeling experiments in which five groups except the control group were subjected to equal amounts of ultraviolet irradiation. Each group of mice had free access to food and water, and four experimental groups of mice were gavaged 2h prior to each exposure at a dose of 100 mg/kg. Irradiating the unhaired mouse at about 30cm under an ultraviolet light source at UVA intensity of 184.3mW/cm²UVB intensity of 78.2mW/cm². Irradiating for 2 hours a day, twice a week and 2-3 days at intervals until molding is completed. Mice after cbs-803 skin tester testing were sacrificed by week 8 and Col I/Col III determined.

ColI/Col III calculation: a1.5 cm square skin on the same back part is taken and placed in precooled normal saline for rinsing, subcutaneous tissues and connective tissues are removed, the skin is wiped and weighed, 9 times volume of precooled normal saline is added, 10% skin homogenate is prepared, and the content of type I collagen and the content of type III collagen in the skin homogenate are determined by an enzyme-linked immunosorbent assay.

The experimental results are as follows

(1) Compared with a blank group, the back skin of the model group mouse has large mild erythema, rough skin texture, obvious wrinkles, dull color and luster, poor elasticity and a large amount of scurf phenomenon on part; the success of molding is demonstrated. In 4 experimental groups using different elastin oligopeptides, only a small amount of erythema appears on the back skin of the mouse, the skin texture is fine and elastic, the color is good, and the experimental groups are superior to model groups and close to blank groups, which shows that the elastin oligopeptides can prevent and improve the skin photoaging of the mouse.

(2) The basic condition of the dorsal skin and Col I/ColIII of each group of mice are shown in tables 1 and 2 below.

TABLE 1 basic dorsal skin conditions in six groups of mice

Group of	Oil content%	Water content%	Pore mm	Elasticity%	Collagen protein fiber%
						Control group	7.3±0.6	17.2±0.5	0.041	56±4	23.1±0.4
Model set	12.6±0.3^##	10.7±0.4^##	0.079	41±3^##	19.2±0.5^##
						Model + example 1 protein peptide	8.5±0.5^**	15.7±0.8^**	0.061	52±5^*	22.2±0.4^**
Model + comparative example protein peptide	10.1±0.6^##**	12.1±0.5^##*	0.063	49±4	20.9±0.5^##*
						Model + example 2 protein peptides	8.3±0.3^**	16.1±0.6^**	0.059	51±3^*	22.4±0.3^**
Model + example 3 protein peptides	8.6±0.6^**	15.5±0.4^#**	0.059	51±5^*	22.1±0.5^**

Note: # denotes P < 0.05% compared to blank; # indicates P < 0.01% compared to blank;

p < 0.05% compared to the blank group; p < 0.01% compared to the blank group. The same applies below.

Through ultraviolet irradiation, the mice in the model group have the advantages of increased skin oil secretion, reduced water content, enlarged pores, reduced elasticity and collagen fibers, and very obvious difference (P is less than 0.01%). The skin oil content, moisture content, pore, elasticity and collagen fiber content of the mice of each experimental group added with the elastin peptide are improved to different degrees, and except for comparison, the skin oil content, the moisture content, the pore, the elasticity and the collagen fiber content are very obvious different from those of a model group (P is less than 0.01%), and each numerical value is closer to that of a blank group. Consistent with the apparent morphological observations.

TABLE 2 Col I/Col III of control, model and mice supplemented with respective protein peptides

Group of	Col I	Col III	Col I/Col III
				Control group	20.2±0.3	11.7±0.4	1.73±0.09
Model set	18.0±0.4^##	13.0±0.3^#	1.38±0.07^##
				Model + example 1 protein peptide	19.6±0.3^*	12.6±0.4	1.56±0.08^*
Model + comparative example protein peptide	19.1±0.6	12.6±0.2^#	1.52±0.07^#
				Model + example 2 protein peptides	19.6±0.5^*	12.4±0.3	1.56±0.04^#*
Model + example 3 protein peptides	19.4±0.5^*	12.3±0.6	1.56±0.04^#*

Compared with the blank group, the collagen composition of the back skin of the model group mice is disordered, the type I collagen is reduced, and the type III collagen is increased and has obvious difference (P is less than 0.05 percent). Compared with the model group, each group of Col I added with elastin oligopeptide is increased back (with statistical significance, P is less than 0.05%), Col III is reduced slightly (with no difference in statistics), and other groups except the comparative example group have Col I/Col III which are higher than the model group, have obvious difference (P is less than 0.05%) and are closer to the blank group. Namely, the protein oligopeptide rich in DES and IDS can inhibit or promote the decomposition or secretion of type I and type III collagen, regulate the ratio of Col I/Col III and maintain the collagen fiber structure of skin.

2. DES and IDS-enriched protein oligopeptide in vitro transdermal permeability test

Using Franz diffusion cell method: filling the receiving pool with normal saline and removing air bubbles, fixing the mouse belly skin after unhairing treatment between the sample pool and the receiving pool, fixing the sample pool, placing 1mL of 10% protein oligopeptide normal saline solution rich in DES and IDS in the horny layer of the mouse skin, namely the sample pool, and taking the receiving liquid as normal saline. The rotation speed is 300r/min, the experiment is started at constant temperature (37 +/-0.5) DEG C, the samples are respectively taken for 2h, 4h, 8h, 12h and 24h, and the sampling quantity is 1ml (V) each time₀) And the receiving cell was supplemented with an equal volume of fresh receiving solution, and the concentration of total amino acids in the receiving solution (ρ) was measured at each sampling time point_n). The volume of the receiving pool is 17mL (V), and the effective contact area is 1.54cm²(A)。

Method for preparing DES and IDS-rich protein oligopeptidesCumulative penetration Q_nThe change with time is shown in table 3.

TABLE 3 cumulative permeation per unit area of each protein oligopeptide at time t_n(unit: μ g/cm)²)

Experimental groups	2h	4h	8h	12h	24h
						Comparative example obtained	371	747	1116	1507	2088
Example 1 results	248	412	734	991	1448
						Example 2 results	252	436	753	1002	1497
Example 3 results	196	322	637	786	1009

The results show that the elastin oligopeptide rich in DES and IDS prepared by each example has good transdermal performance and also provides possibility for the use of the elastin oligopeptide in the aspect of external cosmetics.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. A protein oligopeptide rich in desmosine and isodesmosine, which comprises polypeptides, free amino acids and ash;

2. A method for preparing the protein oligopeptide according to claim 1, which comprises:

sequentially soaking the tissue to be extracted in alkali liquor and acid liquor, grinding to 100-200 meshes, carrying out enzymolysis by using alkaline protease and pancreatin, carrying out enzyme deactivation and centrifugation to obtain supernatant, and filtering and nano-filtering to obtain a solution containing protein oligopeptide;

wherein the tissue to be extracted is an arterial duct, ligament, lung, fascia, cartilage or eggshell membrane.

3. The method according to claim 2, wherein the tissue to be extracted is from pig, cattle, sheep, chicken, duck, goose or fish.

4. The production method according to claim 2,

the alkali liquor is NaHCO₃Solution of NaHCO₃The mass fraction of the alkali liquor is 0.5 wt%, and the alkali liquor is soaked for 0.5-2 hours at normal temperature;

5. The method according to claim 2, wherein the mass ratio of the alkaline protease to the pancreatin is 4: 1; the enzymolysis condition is that the pH is 7.8-8.5; enzymolysis is carried out for 5-6 h at 55-60 ℃.

6. The method of claim 2, wherein the centrifugation is performed at 4500r/min for 20 min; the fine filtration process is that the centrifugal supernatant is filtered by filter membranes with the diameter of 10 mu m and 0.45 mu m in turn; the nanofiltration has a molecular weight cutoff of 160Da, a pressure of 0.5MPa and a time of 5 min.

7. The method according to any one of claims 2 to 6, wherein the method further comprises a step of digesting collagen by an enzymatic method after grinding the collagen to a particle size of 100 to 200 mesh and before the enzymatic method by an alkaline protease and a pancreatin; the enzyme for dissolving out the collagen is papain or a special enzyme for hydrolyzing the collagen.

8. The preparation method according to any one of claims 2 to 7, wherein the solution containing the protein oligopeptide is subjected to reduced pressure concentration until the solid content is 35 to 50 percent, and then is subjected to spray drying to obtain the protein oligopeptide; the inlet temperature of the spray drying is 180 ℃ and the outlet temperature is 70 ℃.

9. Use of the protein oligopeptide according to claim 1 or the protein oligopeptide prepared by the preparation method according to any one of claims 2 to 8 in the preparation of food, medicine and/or cosmetics.

10. A food, pharmaceutical and/or cosmetic composition comprising the protein oligopeptide according to claim 1 or the protein oligopeptide produced by the production method according to any one of claims 2 to 8.