CN111944866B - Method for preparing small molecular collagen peptide of yak leather by continuous rotary steaming desolventizing double enzymolysis - Google Patents
Method for preparing small molecular collagen peptide of yak leather by continuous rotary steaming desolventizing double enzymolysis Download PDFInfo
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Abstract
The invention discloses a method for preparing a yak small molecular collagen peptide by continuous rotary steaming desolventizing double enzymolysis, which comprises the steps of (1) pretreatment of yak, (2) homogenate, (3) enzymolysis, (4) centrifugal separation, (5) decolorization, deodorization and desalination, and (6) concentration and drying, and is characterized in that the enzymolysis of the step (3) comprises the following steps: and (3) performing enzymolysis for the first time, performing superfine treatment, and performing enzymolysis for the second time. The invention can greatly shorten the enzymolysis time and improve the process efficiency by combining double enzymolysis and continuous rotary steaming desolventizing, and the prepared yak skin collagen peptide has high purity, no color and smell, small molecular weight and is beneficial to direct absorption of human bodies.
Description
Technical Field
The invention belongs to the technical field of collagen peptide extraction, and particularly relates to a method for preparing a yak skin micromolecular collagen peptide by continuous rotary steaming desolventizing double enzymolysis.
Background
Qinghai is the largest production base of yaks nationwide, accounting for more than one third of the total number of yaks nationwide, and is prevalent as "all of the world yaks". The unique plateau environment enables the yaks to have extremely strong immunity and strong ultraviolet irradiation, enables the yaks to be treasury of sterile resources, and enables the yaks to have unique nutritional value and reliable safety due to the natural stocking mode. Therefore, the "world ridge" is called as a good raw material for extracting collagen peptide, further increases the utilization value of the yak, and reduces the problem of slaughtering waste.
In the existing skin extraction raw materials, deep sea fish is still the main material, collagen peptide prepared by deep sea fish skin is always the main material in the market, and especially imported deep sea cod skin collagen peptide is popular with the masses. Compared with the collagen peptide of the fish skin, most of the collagen peptide of the cow leather in the current market has yellow color and luster, has the smell of cow leather, has a certain influence on the edible mouthfeel, and also influences the quality of the end product as an additive raw material. Because the yield of collagen peptide prepared by the terrestrial vertebrates is lower than that of marine animals, and a great amount of unhydrolyzed scraps are generated in the enzymolysis process, the filtering burden is increased, and the cost for producing small molecular peptide which is favorable for human absorption is quite high, so that many places for producing the cow leather collagen peptide are not improved. Therefore, it is necessary to develop a more green, environment-friendly, low-cost and high-quality efficient extraction method of the cattlehide collagen peptide, which enhances the utilization value of the Qinghai yak and the market status of the cattlehide peptide in China.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a method for preparing the yak skin small molecular collagen peptide by continuous rotary evaporation desolventizing double enzymolysis.
The technical scheme of the invention is as follows.
The method for preparing the small molecular collagen peptide of the yak skin by continuous rotary steaming desolventizing double enzymolysis comprises the steps of (1) pretreatment of the yak skin, (2) homogenate, (3) enzymolysis, (4) centrifugal separation, (5) decolorization, deodorization and desalination, and (6) concentration and drying, and is characterized in that the enzymolysis in the step (3) comprises the following steps:
first enzymolysis: adding pepsin into the yak skin slurry obtained by homogenizing in the step (2), and performing rotary steaming, desolventizing and enzymolysis for 4-6 hours at the vacuum degree of 0.85-0.98 MPa, the rotating speed of 15-60 r/min and the temperature of 35-40 ℃ to obtain a first enzymolysis liquid;
ultra-fine treatment: inactivating enzyme of the first enzymolysis liquid, sieving with a 200-mesh sieve, collecting undersize liquid, and performing superfine treatment by using a nanometer collision machine to obtain the treated undersize liquid with an average particle diameter smaller than 500nm to obtain a nanometer enzymolysis liquid;
and (3) performing enzymolysis for the second time: adjusting the pH value of the nanocrystallization enzymolysis liquid to a proper range, adding a compound enzyme liquid of alkaline protease and serine protease, and performing rotary steaming desolventizing enzymolysis for 2-3 h at the vacuum degree of 0.85-0.98 MPa, the rotating speed of 15-60 r/min and the temperature of 45-55 ℃; the serine protease is any one of trypsin, subtilisin and elastase;
in the steps, the substrate concentration in the enzymolysis process can be maintained at a higher level by rotating vacuum desolventizing during enzymolysis, so that the integral enzymolysis rate is improved, and the enzymolysis time is greatly shortened; in addition, the enzymolysis liquid is treated by a nano collision machine, so that the average particle size of the enzymolysis liquid containing collagen can reach the nano-scale, the binding site of enzyme and substrate can be increased, and the enzymolysis rate is improved.
As a preferable technical scheme, ultrasonic waves are applied simultaneously when the first enzymolysis and the second enzymolysis in the step (3) are performed with rotary steaming desolventizing enzymolysis.
As a specific technical scheme, the pretreatment of the yak skin in the step (1) is specifically as follows: weighing fresh yak skin, dehairing, cutting into pieces, adding sodium carbonate with the mass concentration of 3% -5% into the fresh yak skin at room temperature according to the mass ratio of 1:3-7 kg/kg, soaking for 6-8 hours, cleaning the yak skin to be neutral by clean water after the soaking is finished, adding the yak skin into salt with the mass concentration of 3% -5% according to the mass ratio of 1:3-7 kg/kg, soaking for 6-8 hours again, and cleaning the yak skin by clean water to be neutral after the soaking is finished.
As a specific technical scheme, the step (2) of homogenizing specifically comprises the following steps: adding 0.05M acetic acid solution into pretreated yak skin according to the mass ratio of 1:4-8 kg/kg, and fully homogenizing by using a colloid mill to obtain yak skin homogenate.
As a specific technical scheme, the centrifugal separation in the step (4) is specifically as follows: and (3) boiling the enzymolysis liquid after the enzymolysis in the step (3) at a high temperature to inactivate enzyme and sterilize, centrifuging at 8000-12000 rpm to remove residues, and micro-filtering to obtain the collagen peptide enzymolysis liquid.
As a specific technical scheme, the decoloring, deodorizing and desalting steps (5) specifically comprise: adding 0.1-0.3% m/v active carbon into the collagen peptide enzymolysis liquid obtained after centrifugal separation in the step (4), stirring for 2-3 h at the room temperature of 40-80 r/min, filtering to remove the active carbon, and further decoloring, desalting and deodorizing by an ion exchange resin column.
As a specific technical scheme, the yak skin in the step (1) is Qinghai-Tidamu basin yak skin.
As a specific technical scheme, the compound enzyme solution is elastase and alkaline protease, and the elastase is introduced in the second enzymolysis to further degrade collagen fibers and collagen expansion, so that the compound enzyme solution has a synergistic effect in the enzymolysis process of the alkaline protease, and the second enzymolysis is greatly promoted, so that the yak skin is fully hydrolyzed.
As a specific technical scheme, pepsin used in the first enzymolysis in the step (3) is food-grade protease extracted from animal mucosa, and the enzyme activity is 10 ten thousand; the alkaline protease used in the second enzymolysis in the step (3) is food-grade protease extracted from bacillus licheniformis, the enzyme activity is 20 ten thousand, the elastase is food-grade protease extracted from pancreas, the enzyme activity is 10 ten thousand, and the optimal enzymolysis effect is achieved by the cooperation of the two enzymes.
As a specific technical scheme, the activated carbon is 200-mesh wood activated carbon, and the ion exchange resin adopts strong acid styrene cation exchange resin and macroporous acrylic weak base anion exchange resin.
As a specific technical scheme, in the yak skin collagen peptide prepared by the method, the content of collagen peptide with the molecular weight of less than 3000Da is more than 95%, the content of oligopeptide with the molecular weight of less than 2000Da is more than 90%, and the content of oligopeptide with the molecular weight of less than 1000Da is more than 70%.
The beneficial effects of the invention are as follows:
1. the invention adopts fresh yak leather in Qinghai-Tidamu basin as raw material, has natural material advantage, and greatly reduces the influence of fat and foreign protein on products and subsequent processes by adopting pretreatment of sodium carbonate and salt.
2. The invention selects colloid mill to carry out homogenization treatment on yak skin, fully crushes the yak skin, and adds the double enzymolysis technology of the invention, the hydrolysis process only comprises acetic acid and NaOH which maintain the enzymolysis pH environment, no special buffer system is needed, after pepsin is used for extracting collagen, elastase is introduced in the second enzymolysis to further degrade collagen fiber and expanded collagen, the enzymolysis process of alkaline protease has synergistic effect, so that the enzymolysis of alkaline protease is greatly promoted, the yak skin is fully hydrolyzed, and only trace crushed yak hair remains in the residual residues, thus greatly reducing the filtering burden. In addition, the invention combines double enzymolysis with continuous rotary steaming desolventizing, which can greatly shorten enzymolysis time and improve process efficiency.
3. According to the invention, the double enzymolysis of pepsin and complex enzyme solution is adopted, ultrafiltration separation is not carried out, and the content of collagen peptide with molecular weight less than 3000Da is more than 95%, the content of oligopeptide with molecular weight less than 2000Da is more than 90%, and the content of oligopeptide with molecular weight less than 1000Da is more than 70%, so that the product is completely beneficial to direct absorption of human body, the production cost is greatly reduced, and the production process is simplified.
4. The invention combines 200-mesh wood activated carbon and anion-cation resin to achieve the best decoloring, desalting and deodorizing effects, can enhance the quality of the yak skin collagen peptide and has better taste.
5. The yak skin collagen Bai Tai obtained by the method is high in purity, colorless and odorless, has obvious advantages compared with most of cow leather collagen peptide products in the market, can be widely applied to foods, health-care products and cosmetics, and does not influence the quality of the terminal products.
Drawings
FIG. 1 is a 12% SDS-PAGE gel electrophoresis analysis chart of a yak skin collagen peptide prepared by the method of example 3, wherein lane 1 is a collagen sample extracted by pepsin in the first enzymolysis, and lane 2 is a yak skin collagen peptide sample obtained by enzymolysis of a complex enzyme solution in the second enzymolysis;
FIG. 2 is a molecular size exclusion chromatogram of a yak skin collagen peptide prepared by the method of example 3, wherein a single peak in the chromatogram is the retention time of a standard peptide, and the molecular weight distribution of the collagen peptide;
FIG. 3 is a mass spectrum of a matrix assisted laser Desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) of a yak skin collagen peptide prepared by the method of example 3.
Detailed Description
The present invention will be further described with reference to the following specific embodiments, but it should be noted that the following embodiments are merely illustrative of the present invention, and the scope of the present invention is not limited thereto, and all equivalents thereof by those skilled in the art to which the present invention pertains fall within the scope of the present invention.
Example 1
The method for preparing the small molecular collagen peptide of the yak skin by continuous rotary steaming desolventizing double enzymolysis comprises the following specific steps of (1) pretreatment of the yak skin, (2) homogenate, (3) enzymolysis, (4) centrifugal separation, (5) decolorization, deodorization and desalination, and (6) concentration and drying:
(1) Pretreatment: weighing 50g of fresh yak skin, dehairing, cutting into blocks with the mass ratio of 1X 1cm, adding sodium carbonate with the mass concentration of 5% into the blocks at room temperature according to the mass ratio of 1:5kg/kg, soaking for 8 hours, cleaning the yak skin to be neutral by clean water after soaking, adding the yak skin into salt with the mass concentration of 5% according to the mass ratio of 1:5kg/kg, soaking for 8 hours again, and cleaning the yak skin to be neutral by clean water after soaking, so as to achieve the purposes of degreasing and impurity removal;
(2) Homogenizing: adding 0.05M acetic acid solution into pretreated yak skin according to the mass ratio of 1:5kg/kg, and fully homogenizing by using a colloid mill to obtain yak skin homogenate;
(3) Enzymolysis:
first enzymolysis: adding 1.5g of pepsin with the enzyme activity of 10 ten thousand into the yak skin slurry obtained by homogenizing in the step (2), transferring the mixture into rotary evaporation equipment, and performing rotary evaporation, desolventizing and enzymolysis for 6 hours at the temperature of 35-40 ℃ under the vacuum degree of 0.85-0.98 MPa and the rotating speed of 20r/min to obtain a first enzymolysis solution;
ultra-fine treatment: inactivating enzyme of the first enzymolysis liquid, sieving with a 200-mesh sieve, collecting undersize liquid, and performing superfine treatment by using a nanometer collision machine to obtain the treated undersize liquid with an average particle diameter smaller than 500nm to obtain a nanometer enzymolysis liquid;
and (3) performing enzymolysis for the second time: adjusting the pH value of the nanocrystallization enzymolysis liquid to 9 by adopting NaOH, adding 0.3g of alkaline protease with the enzyme activity of 20 ten thousand and 0.03g of elastase with the enzyme activity of 10 ten thousand, then transferring the mixture into rotary evaporation equipment, and performing rotary evaporation, desolventizing and enzymolysis for 3 hours at the temperature of 50 ℃ under the vacuum degree of 0.85-0.98 MPa and the rotating speed of 60 r/min;
(4) And (3) centrifugal separation: and (3) boiling the enzymolysis liquid after the enzymolysis in the step (3) at a high temperature for 30min to perform enzyme deactivation and sterilization, centrifuging at 10000rpm to remove residues, and performing microfiltration to obtain the collagen peptide enzymolysis liquid.
(5) Decolorization, deodorization and desalination: adding 1.5g of 200-mesh wood activated carbon into the collagen peptide enzymatic hydrolysate obtained after centrifugal separation in the step (4), stirring for 2-3 hours at room temperature at 60r/min, filtering to remove the activated carbon, and further decoloring, desalting and deodorizing by an ion exchange resin column.
(6) Concentrating and drying: and concentrating the enzymolysis solution treated in the previous step through a falling film, and performing spray drying to obtain colorless and odorless yak skin collagen peptide, wherein the yield of the yak skin collagen peptide is 15.6%.
Example 2
The method for preparing the small molecular collagen peptide of the yak skin by continuous rotary steaming desolventizing double enzymolysis comprises the following specific steps of (1) pretreatment of the yak skin, (2) homogenate, (3) enzymolysis, (4) centrifugal separation, (5) decolorization, deodorization and desalination, and (6) concentration and drying:
(1) Pretreatment: weighing 500g of fresh yak skin, dehairing, cutting into blocks with the mass ratio of 1X 1cm, adding sodium carbonate with the mass concentration of 3% into the blocks at room temperature according to the mass ratio of 1:5kg/kg, soaking for 8 hours, cleaning the yak skin to be neutral by clean water after soaking, adding the yak skin into salt with the mass concentration of 3% according to the mass ratio of 1:5kg/kg, soaking for 8 hours again, and cleaning the yak skin to be neutral by clean water after soaking, so as to achieve the purposes of degreasing and impurity removal;
(2) Homogenizing: adding 0.05M acetic acid solution into pretreated yak skin according to the mass ratio of 1:5kg/kg, and fully homogenizing by using a colloid mill to obtain yak skin homogenate;
(3) Enzymolysis:
first enzymolysis: adding 15g of pepsin with the enzyme activity of 10 ten thousand into the yak skin slurry obtained by homogenizing in the step (2), transferring the mixture into rotary evaporation equipment, and performing rotary evaporation, desolventizing and enzymolysis for 5 hours at the vacuum degree of 0.85-0.98 MPa, the rotating speed of 30r/min and the temperature of 35-40 ℃ to obtain a first enzymolysis solution;
ultra-fine treatment: inactivating enzyme of the first enzymolysis liquid, sieving with a 200-mesh sieve, collecting undersize liquid, and performing superfine treatment by using a nanometer collision machine to obtain the treated undersize liquid with an average particle diameter smaller than 500nm to obtain a nanometer enzymolysis liquid;
and (3) performing enzymolysis for the second time: regulating the pH value of the nanocrystallization enzymolysis liquid to 9 by adopting NaOH, adding 3g of alkaline protease with the enzyme activity of 20 ten thousand and 0.3g of elastase with the enzyme activity of 10 ten thousand, then transferring the mixture into rotary evaporation equipment, and performing rotary evaporation, desolventizing and enzymolysis for 2.5 hours at the temperature of 50 ℃ and the rotation speed of 40r/min under the vacuum degree of 0.85-0.98 MPa;
(4) And (3) centrifugal separation: boiling the enzymolysis solution obtained after the enzymolysis in the step (3) at high temperature for 30min to inactivate enzyme and sterilize, centrifuging at 10000rpm to remove residues, and micro-filtering to obtain collagen peptide enzymolysis solution;
(5) Decolorization, deodorization and desalination: adding 1.5g of 200-mesh wood activated carbon into the collagen peptide enzymatic hydrolysate obtained after centrifugal separation in the step (4), stirring for 2-3 hours at room temperature for 60r/min, filtering to remove the activated carbon, and further decoloring, desalting and deodorizing by a pre-packaged styrene cation exchange resin and macroporous acrylic weak base anion exchange resin column;
(6) Concentrating and drying: and concentrating the enzymolysis solution treated in the previous step through a falling film, and performing spray drying to obtain colorless and odorless yak skin collagen peptide, wherein the yield of the yak skin collagen peptide is 15.2%.
Example 3
The method for preparing the small molecular collagen peptide of the yak skin by continuous rotary steaming desolventizing double enzymolysis comprises the following specific steps of (1) pretreatment of the yak skin, (2) homogenate, (3) enzymolysis, (4) centrifugal separation, (5) decolorization, deodorization and desalination, and (6) concentration and drying:
(1) Pretreatment: weighing 20kg of fresh yaks, unhairing, cutting into blocks with the mass ratio of 1X 1cm, adding sodium carbonate with the mass concentration of 5% into the blocks at room temperature according to the mass ratio of 1:5kg/kg, soaking for 8 hours, cleaning the yaks to be neutral by clean water after the soaking is finished, adding the yaks into salt with the mass concentration of 5% according to the mass ratio of 1:5kg/kg, soaking for 8 hours again, and cleaning the yaks to be neutral by clean water after the soaking is finished so as to achieve the purposes of degreasing and impurity removal;
(2) Homogenizing: adding 0.05M acetic acid solution into pretreated yak skin according to the mass ratio of 1:5kg/kg, and fully homogenizing by using a colloid mill to obtain yak skin homogenate;
(3) Enzymolysis:
first enzymolysis: adding 0.6kg of pepsin with the enzyme activity of 10 ten thousand into the yak skin slurry obtained by homogenizing in the step (2), transferring the mixture into rotary evaporation equipment, and performing rotary evaporation, desolventizing and enzymolysis for 4 hours at the temperature of 35-40 ℃ under the vacuum degree of 0.85-0.98 MPa and the rotating speed of 20r/min to obtain a first enzymolysis solution;
ultra-fine treatment: inactivating enzyme of the first enzymolysis liquid, sieving with a 200-mesh sieve, collecting undersize liquid, and performing superfine treatment by using a nanometer collision machine to obtain the treated undersize liquid with an average particle diameter smaller than 500nm to obtain a nanometer enzymolysis liquid;
and (3) performing enzymolysis for the second time: adjusting the pH value of the nanocrystallization enzymolysis liquid to 9 by adopting NaOH, adding 0.12kg of alkaline protease with the enzyme activity of 20 ten thousand and 0.012kg of elastase with the enzyme activity of 10 ten thousand, then transferring the mixture into rotary evaporation equipment, and performing rotary evaporation, desolventizing and enzymolysis for 2 hours at the temperature of 50 ℃ under the vacuum degree of 0.85-0.98 MPa and the rotating speed of 15-60 r/min;
(4) And (3) centrifugal separation: and (3) boiling the enzymolysis liquid after the enzymolysis in the step (3) at a high temperature for 30min to perform enzyme deactivation and sterilization, centrifuging at 10000rpm to remove residues, and performing microfiltration to obtain the collagen peptide enzymolysis liquid.
(5) Decolorization, deodorization and desalination: adding 0.1-0.3% m/v of wood activated carbon (200 meshes) into the collagen peptide enzymolysis liquid obtained after centrifugal separation in the step (4), stirring for 2-3 h at room temperature for 60r/min, filtering to remove the activated carbon, and further decoloring, desalting and deodorizing by an ion exchange resin column.
(6) Concentrating and drying: and concentrating the enzymolysis solution treated in the last step through a falling film, and performing spray drying to obtain colorless and odorless yak skin collagen peptide, wherein the yield of the yak skin collagen peptide is 13.2%.
Example 4
The characteristic analysis of the collagen peptide was carried out by taking the yak skin collagen peptide prepared by the method of example 3 of the present invention as a sample.
1) As can be seen from fig. 1, the yak skin collagen extracted by the first enzymolysis, i.e. pepsin enzymolysis, in lane 1 is collagen with a complete triple helix structure; in lane 2, collagen peptide is obtained by further enzymolysis of compound enzyme solution, namely enzymolysis of collagen protein.
2) As can be seen from fig. 2, according to the detection method of molecular weight distribution in food safety national standard GB/T22729-2008, the result shows that the proportion of collagen peptide with relative molecular mass less than 10000Da in the yak skin collagen peptide prepared in example 3 is completely more than 90%, reaching the national standard. In addition, in the yak skin collagen peptide prepared by the preparation method, the content of the collagen peptide with the molecular weight of less than 3000Da is more than 95 percent, the content of the oligopeptide with the molecular weight of less than 2000Da is more than 90 percent, and the content of the oligopeptide with the molecular weight of less than 1000Da is more than 70 percent, so that the prepared product is beneficial to direct absorption of human bodies.
3) As can be seen from FIG. 3, the MALDI-TOF-MS mass spectrum identification result shows that the molecular weight of the yak skin collagen peptide prepared in the example 3 is mainly concentrated below 3000 Da.
Claims (9)
1. The method for preparing the small molecular collagen peptide of the yak skin by continuous rotary steaming desolventizing double enzymolysis is characterized by comprising the steps of (1) pretreatment of the yak skin, (2) homogenate, (3) enzymolysis, (4) centrifugal separation, (5) decolorization, deodorization and desalination, and (6) concentration and drying, wherein the enzymolysis of the step (3) comprises the following steps:
first enzymolysis: adding pepsin into the yak skin slurry obtained by homogenizing in the step (2), and performing rotary steaming, desolventizing and enzymolysis at the vacuum degree of 0.85-0.98 MPa, the rotating speed of 15-60 r/min and the temperature of 35-40 ℃ for 4h to obtain a first enzymolysis liquid;
ultra-fine treatment: inactivating enzyme of the first enzymolysis liquid, sieving with a 200-mesh sieve, collecting undersize liquid, and performing superfine treatment by using a nanometer collision machine to obtain the treated undersize liquid with an average particle size smaller than 500nm to obtain nanocrystallized enzymolysis liquid;
and (3) performing enzymolysis for the second time: adjusting the pH value of the nanocrystallization enzymolysis liquid to 9, adding alkaline protease and elastase, and then performing rotary evaporation desolventizing enzymolysis at the vacuum degree of 0.85-0.98 MPa, the rotating speed of 15-60 r/min and the temperature of 50 ℃ for 2 h.
2. The method for preparing the small molecular collagen peptide of the yak skin by continuous rotary steaming desolventizing double enzymolysis according to claim 1, wherein the pretreatment of the yak skin in the step (1) is specifically as follows: weighing fresh yak skin, dehairing, cutting into pieces, adding sodium carbonate with the mass concentration of 3% -5% into the fresh yak skin at room temperature according to the mass ratio of 1:3-7 kg/kg, soaking for 6-8 hours, cleaning the yak skin to be neutral by clean water after the soaking is finished, adding the yak skin into salt with the mass concentration of 3% -5% according to the mass ratio of 1:3-7 kg/kg, soaking for 6-8 hours again, and cleaning the yak skin by clean water to be neutral after the soaking is finished.
3. The method for preparing the small molecular collagen peptide of the yak skin by continuous rotary steaming desolventizing double enzymolysis according to claim 1, wherein the step (2) homogenate is specifically: adding 0.05M acetic acid solution into pretreated yak skin according to the mass ratio of 1:4-8 kg/kg, and fully homogenizing by using a colloid mill to obtain yak skin homogenate.
4. The method for preparing the small molecular collagen peptide of the yak skin by continuous rotary evaporation, desolventizing and double enzymolysis according to claim 1, wherein the centrifugal separation in the step (4) is specifically: and (3) boiling the enzymolysis liquid after the enzymolysis in the step (3) at a high temperature to inactivate enzyme and sterilize, centrifuging at 8000-12000 rpm to remove residues, and micro-filtering to obtain the collagen peptide enzymolysis liquid.
5. The method for preparing the yak small molecule collagen peptide by continuous rotary steaming desolventizing double enzymolysis according to claim 1, wherein the decoloring, deodorizing and desalting in the step (5) is specifically as follows: adding 0.1% -0.3% m/v of activated carbon into the collagen peptide enzymolysis liquid obtained after centrifugal separation in the step (4), stirring for 2-3 hours at the room temperature of 40-80 r/min, filtering to remove the activated carbon, and further decoloring, desalting and deodorizing by an ion exchange resin column.
6. The method for preparing the yak small molecule collagen peptide by continuous rotary steaming desolventizing double enzymolysis according to claim 1, wherein the method comprises the following steps: the yak skin in the step (1) is Qinghai-chaidamu basin yak skin.
7. The method for preparing the yak small molecule collagen peptide by continuous rotary steaming desolventizing double enzymolysis according to claim 1, wherein the method comprises the following steps: the pepsin used for the first enzymolysis in the step (3) is food-grade protease extracted from animal mucosa; the alkaline protease used in the second enzymolysis in the step (3) is food-grade protease extracted from bacillus licheniformis, and the elastase is food-grade protease extracted from pancreas.
8. The method for preparing the yak small molecule collagen peptide by continuous rotary steaming desolventizing double enzymolysis according to claim 5, wherein the method comprises the following steps: the activated carbon is 200-mesh wood activated carbon, and the ion exchange resin adopts strong acid styrene cation exchange resin and macroporous acrylic weak base anion exchange resin.
9. The method for preparing the yak skin small molecule collagen peptide by continuous rotary steaming desolventizing double enzymolysis according to any one of claims 1 to 8, which is characterized by comprising the following steps: in the small molecular collagen peptide of the yak skin prepared by the method, the content of the collagen peptide with the molecular weight of less than 3000Da is more than 95 percent, the content of the oligopeptide with the molecular weight of less than 2000Da is more than 90 percent, and the content of the oligopeptide with the molecular weight of less than 1000Da is more than 70 percent.
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| CN112569347B (en) * | 2020-12-30 | 2023-09-22 | 青海瑞肽生物科技有限公司 | Application of yak collagen peptide in hypoglycemic drugs or hypoglycemic health-care foods |
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| CN117736307B (en) * | 2024-02-19 | 2024-06-07 | 北京盛美诺生物技术有限公司 | III type small molecule collagen peptide capable of promoting III type and IV type collagen secretion and preparation method thereof |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008074846A (en) * | 2006-08-25 | 2008-04-03 | Nippon Meat Packers Inc | Elastin decomposition peptide and method for preparing elastin and its zymolytic peptide |
| CN102212107A (en) * | 2011-04-12 | 2011-10-12 | 江南大学 | Rice protein polypeptide and preparation method thereof |
| CN105695548A (en) * | 2016-03-30 | 2016-06-22 | 蔡庭守 | Preparation method of donkey-hide gelatin small molecular peptide |
| CN106011208A (en) * | 2016-06-29 | 2016-10-12 | 肖建喜 | Method for preparing small-molecular weight collagen active peptide through enzymolysis of yak bone and skin |
| WO2018014841A1 (en) * | 2016-07-19 | 2018-01-25 | 华南生物医药研究院 | Self-assembled collagen and preparation method therefor |
| CN107653291A (en) * | 2017-11-15 | 2018-02-02 | 西藏央金生态农牧科技有限公司 | The standby method for hiding Yak-skin Gelatin original albumen and collagen polypeptide of multi-step enzyme method coordinate system |
| CN109295145A (en) * | 2018-10-31 | 2019-02-01 | 西藏央金生态农牧科技有限公司 | A kind of preparation method of Ultra-low molecular weight yak collagen peptide |
| CN110229862A (en) * | 2019-06-21 | 2019-09-13 | 中国科学院西北高原生物研究所 | A kind of Yak-skin Gelatin preparation method promoting platelet activation |
| CN110592053A (en) * | 2019-09-29 | 2019-12-20 | 河南新仰韶生物科技有限公司 | Collagen hydrolysis complex enzyme, protein oligopeptide and preparation method thereof |
| CN110628857A (en) * | 2019-10-29 | 2019-12-31 | 南宁学院 | Method for extracting collagen from cow leather |
| CN111363772A (en) * | 2020-04-08 | 2020-07-03 | 平凉市华科生物技术有限公司 | Method for preparing collagen peptide by hydrolyzing bovine bone and collagen peptide thereof |
-
2020
- 2020-07-31 CN CN202010775142.0A patent/CN111944866B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008074846A (en) * | 2006-08-25 | 2008-04-03 | Nippon Meat Packers Inc | Elastin decomposition peptide and method for preparing elastin and its zymolytic peptide |
| CN102212107A (en) * | 2011-04-12 | 2011-10-12 | 江南大学 | Rice protein polypeptide and preparation method thereof |
| CN105695548A (en) * | 2016-03-30 | 2016-06-22 | 蔡庭守 | Preparation method of donkey-hide gelatin small molecular peptide |
| CN106011208A (en) * | 2016-06-29 | 2016-10-12 | 肖建喜 | Method for preparing small-molecular weight collagen active peptide through enzymolysis of yak bone and skin |
| WO2018014841A1 (en) * | 2016-07-19 | 2018-01-25 | 华南生物医药研究院 | Self-assembled collagen and preparation method therefor |
| CN107653291A (en) * | 2017-11-15 | 2018-02-02 | 西藏央金生态农牧科技有限公司 | The standby method for hiding Yak-skin Gelatin original albumen and collagen polypeptide of multi-step enzyme method coordinate system |
| CN109295145A (en) * | 2018-10-31 | 2019-02-01 | 西藏央金生态农牧科技有限公司 | A kind of preparation method of Ultra-low molecular weight yak collagen peptide |
| CN110229862A (en) * | 2019-06-21 | 2019-09-13 | 中国科学院西北高原生物研究所 | A kind of Yak-skin Gelatin preparation method promoting platelet activation |
| CN110592053A (en) * | 2019-09-29 | 2019-12-20 | 河南新仰韶生物科技有限公司 | Collagen hydrolysis complex enzyme, protein oligopeptide and preparation method thereof |
| CN110628857A (en) * | 2019-10-29 | 2019-12-31 | 南宁学院 | Method for extracting collagen from cow leather |
| CN111363772A (en) * | 2020-04-08 | 2020-07-03 | 平凉市华科生物技术有限公司 | Method for preparing collagen peptide by hydrolyzing bovine bone and collagen peptide thereof |
Non-Patent Citations (3)
| Title |
|---|
| Effect of Orally Administered Collagen Peptides from Bovine Bone on Skin Aging in Chronologically Aged Mice;Hongdong Song等;《nutrients》;20171103;第1-14页 * |
| 弹性蛋白酶提取牛蛙皮胶原蛋白的工艺优化;杨远帆等;《中国食品学报》;20131031(第10期);第66-72页 * |
| 碱性蛋白酶提取牦牛皮胶原蛋白的工艺研究;陈渭;《青海大学学报》;20170831;第35卷(第4期);第71-75页 * |
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