CN111548409A - Extraction process of animal fresh skin collagen polypeptide - Google Patents
Extraction process of animal fresh skin collagen polypeptide Download PDFInfo
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- CN111548409A CN111548409A CN202010434058.2A CN202010434058A CN111548409A CN 111548409 A CN111548409 A CN 111548409A CN 202010434058 A CN202010434058 A CN 202010434058A CN 111548409 A CN111548409 A CN 111548409A
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- extraction
- enzymolysis
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Abstract
The invention relates to an extraction process of animal fresh skin collagen polypeptide, which comprises the following steps: pretreating animal skin, performing ultrasonic extraction, performing high-pressure homogenization and secondary bionic enzymolysis to collect enzymolysis liquid. The invention simulates the environment of the human digestive tract to establish an enzymolysis reactor to carry out secondary bionic enzymolysis on the collagen extracting solution so as to obtain the collagen peptide of the animal fresh skin. The extraction process can release collagen of the skin as effectively as possible, and can change the collagen from a protein molecule tertiary-quaternary structure into a primary protein peptide chain structure through a physical high-pressure shearing technology. The extraction process can not only improve production efficiency, but also improve production yield. The extraction process can produce collagen peptide which is more in line with the absorption and biological effects of human bodies, and improves the absorption utilization rate of the collagen peptide; the biological activity of the effective components of the product is ensured, the application range and the use effect of the product are expanded, and the worry of toxic and side effects is avoided.
Description
Technical Field
The invention relates to the field of extraction of collagen peptides, in particular to an extraction process of collagen polypeptide from animal fresh skin.
Background
The collagen peptide has high health care effect because the collagen peptide is a micromolecular protein, compared with common collagen, the collagen peptide has higher absorption rate, the absorption utilization rate of edible collagen peptide can reach 100 percent, and the collagen peptide can be actively absorbed by human body without barriers and can be fully utilized by body tissues. After being fully absorbed and utilized by a human body, the collagen can be synthesized into skin collagen to play a certain beautifying role, and the collagen has very good hydrophilicity and can provide a plurality of whitening, moisturizing and nourishing effects for the skin, so that the aims of improving the skin quality and beautifying are fulfilled; the collagen can promote the formation of bones and strengthen the collagen structure under the low calcium level, thereby improving the bone strength and achieving the effect of preventing osteoporosis; the collagen peptide keeps the muscles and bones soft and elastic, strengthens the lubrication degree of the muscles and bones during movement and friction, reduces the incidence rate of arthritis and can avoid joint degeneration; the collagen can improve anxiety and insomnia states and treat neurasthenia; the collagen is helpful for strengthening the functions of various tissues and organs and improving the immunity of the human body.
The protein (food or medicine) ingested by people needs to be digested and enzymolyzed into small peptide and amino acid by digestive enzyme in the human digestive system, namely gastric juice and intestinal juice, and then absorbed and utilized by the organism to play the biological effect. The animal skin glue such as donkey-hide gelatin, oxhide gelatin and the like which are used as medicine and food are generally melted and blended for taking. But has the disadvantages of difficult digestion and absorption, especially for patients with weakness of the spleen and the stomach, not only difficult absorption but also a certain digestive burden. The problem to be solved at present is to provide a method for developing a small molecular collagen peptide which is suitable for human body to absorb and can exert the pharmaceutical effect of the collagen peptide and accelerate the metabolism of the collagen in vivo by effectively utilizing animal skin resources.
Disclosure of Invention
In order to solve the problems, the invention provides an extraction process of collagen polypeptide from animal fresh skin, which comprises the steps of pretreating animal fresh skin (donkey skin, cow skin, pigskin, horse skin and the like), and extracting collagen and collagen peptide from the animal skin by using a low-temperature ultrasonic extraction and high-pressure homogenization linkage technology; and then simulating the human digestive tract environment to establish an enzymolysis reactor to carry out secondary bionic enzymolysis on the collagen extracting solution to obtain the animal fresh skin micromolecule collagen peptide. The method not only innovates the extraction process technology of the collagen peptide, saves the extraction time, improves the production efficiency, improves the quality and the labor efficiency of the collagen peptide, but also promotes the technical progress and the industrialization process of the collagen peptide industry to a great extent. In addition, the collagen peptide obtained by the secondary bionic enzymolysis technology is easy to absorb and has good biological effect.
In order to achieve the purpose of the invention, the invention provides an extraction process of animal skin collagen peptide, which is characterized by comprising the following steps: ultrasonic extraction, high-pressure homogenization and secondary bionic enzymolysis.
The invention has the beneficial effects that:
firstly, the method of the invention applies an ultrasonic-high pressure homogenization linkage technology, so that the collagen of the animal skin can be released as effectively as possible, the linkage high pressure homogenization technology can release the collagen more effectively, and simultaneously, the collagen can be changed into nano-scale small molecular peptides from protein molecules through a physical high pressure shearing technology, and is changed into a primary protein peptide chain structure from a protein molecule tertiary and quaternary structure through the physical high pressure shearing technology. The technology can not only improve the production efficiency, but also improve the production yield.
Secondly, the method simulates the physiological environment of the stomach and the intestine of a human, and prepares the collagen peptide of the animal skin by adopting a two-stage bionic enzymolysis technology, so that the collagen peptide which is more in line with the absorption and biological effects of the human body can be produced, and the absorption utilization rate of the collagen peptide is improved.
Thirdly, the animal skin collagen peptide prepared by the method not only ensures the bioactivity of the effective components of the product, but also expands the application range and the use effect of the product and avoids worrying about toxic and side effects.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a technical flow chart of the extraction process of the collagen polypeptide of the animal fresh skin.
FIG. 2 shows chromatograms of L-hydroxyproline, glycine, alanine, L-proline controls.
FIG. 3 shows chromatogram maps and detection results of L-hydroxyproline, glycine, alanine and L-proline in donkey skin collagen peptide.
FIG. 4 shows chromatogram maps and detection results of L-hydroxyproline, glycine, alanine and L-proline in bovine collagen peptide.
FIG. 5 shows chromatogram maps and detection results of L-hydroxyproline, glycine, alanine and L-proline in pigskin-derived collagen peptide.
FIG. 6 shows chromatogram maps and detection results of L-hydroxyproline, glycine, alanine and L-proline in horse skin collagen peptide.
FIG. 7 shows the SDS-PAGE result of the donkey skin collagen peptide small molecular polypeptide, wherein: lane 1 is marine fish collagen peptide powder; lane 2 is E.coli polypeptide powder; lane 3 is the collagen peptide derived from donkey skin prepared in example 1; lane 4 is protein Marker (Marker).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly shown, embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.
As shown in fig. 1, the extraction process of the animal fresh skin collagen peptide provided by the invention comprises the following steps: pretreating fresh animal skin (cleaning, defatting, mincing), ultrasonic extracting, homogenizing under high pressure, extracting and separating succus Myristicae, primarily concentrating and extracting foam, performing secondary bionic enzymolysis, centrifuging, collecting enzymolysis solution, filtering, and packaging.
The invention is realized by the following technical scheme:
an extraction process of animal skin collagen peptide, which is characterized by comprising the following steps: ultrasonic extraction, high-pressure homogenization and secondary bionic enzymolysis.
In a preferred embodiment of the present invention, the process for extracting animal skin collagen peptide further comprises one or more steps selected from the group consisting of: pretreating animal skin, extracting and separating glue juice, primarily concentrating and foam extracting, collecting enzymolysis liquid, filtering and filling; preferably, the animal skin is animal hide; preferably, the animal skin is selected from donkey hide, horse hide, cow hide or pig hide.
In a preferred embodiment of the present invention, the process for extracting animal skin collagen peptide comprises the following steps:
(1) pretreating animal skins;
(2) ultrasonic extraction;
(3) homogenizing under high pressure;
(4) extracting and separating the glue juice;
(5) primary concentration and foam extraction;
(6) performing secondary bionic enzymolysis;
(7) and collecting the enzymolysis liquid.
In a preferred embodiment of the present invention, the process for extracting collagen peptide from animal skin comprises the steps of (1) pretreating animal skin, wherein the step comprises: mincing animal skin to obtain minced animal skin tissue.
In one embodiment of the present invention, skin surface impurities, fascia and fat tissue are removed, and then the animal skin is minced to obtain minced animal skin tissue, i.e., pretreated skin material.
In a specific embodiment of the invention, the animal skin treatment is washed for a plurality of times by using water, preferably purified water, preferably for 2-3 times, then impurities, fascia and fat tissues on the skin surface are removed, and then the animal skin is minced to obtain minced animal skin tissues.
In a preferred embodiment of the present invention, the process for extracting animal skin collagen peptide comprises the following steps: adding water with multiple volumes into the minced animal skin tissue, and extracting in an ultrasonic extractor to obtain tissue fluid after ultrasonic extraction.
In a specific embodiment of the present invention, 3-5 times water, more preferably 3 times water is added to the minced animal skin tissue.
In a specific embodiment of the invention, the low-temperature extraction is carried out in an ultrasonic extractor, and more preferably, the extraction temperature is 2-4 ℃.
In one specific embodiment of the invention, the ultrasonic power is 300-500 w/L.
In one embodiment of the invention, the stirring speed is 1000-1200 r/min.
In a specific embodiment of the invention, the ultrasonic time is 30-50 min.
In a specific embodiment of the invention, the ultrasonic power is 400w/L, the stirring speed is 1000r/min, and the ultrasonic time is 30 min.
In a preferred embodiment of the present invention, the process for extracting animal skin collagen peptide, wherein the step (3) of high pressure homogenization comprises: and (3) carrying out high-pressure homogenization on the tissue fluid obtained in the step (2) after ultrasonic extraction in a high-pressure homogenizer to obtain the homogenized tissue fluid after high-pressure homogenization.
In one embodiment of the invention, high pressure homogenization is performed at low temperature; preferably, the high-pressure homogenizing temperature is 0-10 ℃.
In a specific embodiment of the present invention, the high-pressure homogenization is repeated a plurality of times, preferably 3 to 5 times, and preferably 3 times.
In a particular embodiment of the invention, the homogenizing shear is carried out at a homogenizing pressure of 7000Pa, 5000 Pa and preferably 6000 Pa.
In one embodiment of the invention, the homogenizing shear is carried out at a homogenizing rate of 300-700ml/min, preferably 500 ml/min.
In a preferred embodiment of the present invention, the process for extracting collagen peptide from animal skin comprises the following steps: cooking the homogenized tissue fluid obtained in the step (3) in a cooking kettle to obtain glue juice and a skin tissue; separating the glue juice from the skin tissue by filtration to obtain filtrate and separated skin tissue, and collecting filtrate to obtain glue juice extract.
In one embodiment of the invention, the mixture is cooked in a high-pressure cooking kettle, preferably at 121 ℃, and 0.2MPa for 1.5-2 h, preferably 1.5 h.
In one embodiment of the present invention, the above-mentioned gel juice is separated from the skin tissue by a stepwise filtration method, preferably by a filtration screen of 80 mesh, 120 mesh, 200 mesh, 300 mesh, 400 mesh, or 500 mesh, and the gel juice extract and the separated skin tissue are collected.
In a specific embodiment of the invention, water with multiple volumes, preferably 1-2 times water, more preferably 1.5 times water is added into the separated skin tissue according to the mass-volume ratio of the pretreated skin tissue in the step (1), the extraction and separation of the glue juice are repeatedly carried out for multiple times to obtain filtrate and the separated skin tissue, the filtrate is combined, and the filtrate is collected to obtain the glue juice extracting solution; preferably, the extraction and separation of the glue juice are repeated for 2-5 times, more preferably 3 times.
In a preferred embodiment of the present invention, the process for extracting collagen peptide from animal skin comprises the steps of (5) primarily concentrating and extracting foam comprising: heating and primarily concentrating the glue juice extracting solution collected in the step (4); removing froth generated in the concentration process, namely foam extraction; obtaining the glue juice after the primary concentration and the foam extraction are finished.
In one embodiment of the invention, the stirring is continued during the initial concentration.
In one embodiment of the invention, the foam is extracted by gently lifting the foam along the edge of the pan with a wooden spoon and adding a suitable amount of water, preferably purified water.
In a specific embodiment of the invention, the foam extraction operation is repeated continuously until the glue juice is transparent and no froth is generated, the primary concentration and the foam extraction are finished, and the glue juice after the primary concentration and the foam extraction are finished is obtained.
In a preferred embodiment of the present invention, the process for extracting animal skin collagen peptide, wherein the step (6) of secondary biomimetic enzymatic hydrolysis comprises: and (3) carrying out secondary bionic enzymolysis on the primary concentrated and foam-extracted glue juice obtained in the step (5) to carry out enzymolysis on the glue juice, wherein the secondary bionic enzymolysis comprises the following steps:
I. first-order biological enzymolysis (simulated artificial gastric juice):
and (3) adjusting the pH value of the glue solution after the primary concentration and foam extraction to 1.5-2.5 by using acid or alkali, preferably adjusting the pH value to 2.0, and adding pepsin (10 ten thousand U/g) into the glue solution after the primary concentration and foam extraction according to the volume percentage of 0.5-1.0% of the glue solution after the primary concentration and foam extraction to obtain an enzymatic hydrolysate after primary biological enzymolysis.
In a particular embodiment of the invention, the acid is selected from one or more of hydrochloric acid, phosphoric acid, sulfuric acid, oxalic acid, acetic acid and citric acid, preferably HCl.
In a particular embodiment of the invention, the base is selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate and sodium bicarbonate and triethanolamine, preferably NaOH.
In a particular embodiment of the invention, the pH is adjusted with 2.0mol/L HCl and/or 2mol/L NaOH.
In a particular embodiment of the invention, 0.8% pepsin (10 ten thousand U/g) is added; preferably, enzymolysis is carried out for 2.0 h-2.5 h at 37 ℃ +/-0.5 ℃.
In a specific embodiment of the invention, heating to 90-100 ℃ to inactivate enzyme to obtain enzymolysis liquid after primary biological enzymolysis; preferably, the enzyme is inactivated for 15-20 min, more preferably, the enzyme is inactivated for 15 min.
Secondary biological enzymatic hydrolysis (simulated human factory intestinal fluid):
adjusting the pH value of the enzymatic hydrolysate after the primary biological enzymolysis to 7.5-8.0 by using a buffer solution, preferably adjusting the pH value to 8.0; adding pancreatin (4000U/g) into the pH-adjusted enzymatic hydrolysate after the primary biological enzymolysis according to the volume percentage of 0.5-1.0% of the glue juice after the primary concentration and foam extraction; obtaining enzymolysis liquid after the second-stage biological enzymolysis.
In a particular embodiment of the invention, the buffer is a phosphate buffer.
In a particular embodiment of the invention, 0.5% pancreatin (4000U/g) is added; more preferably, enzymolysis is carried out for 2.0 h-2.5 h at 50 ℃ +/-0.5 ℃.
In a specific embodiment of the invention, the temperature is raised to 90-100 ℃ to inactivate the enzyme, so as to obtain enzymatic hydrolysate after secondary biological enzymolysis; preferably, the enzyme is inactivated for 15-20 min, more preferably, the enzyme is inactivated for 15 min.
In a preferred embodiment of the present invention, the process for extracting collagen peptide from animal skin, wherein the step (7) of collecting the enzymatic hydrolysate comprises centrifuging the enzymatic hydrolysate after the secondary biological enzymolysis, and collecting the supernatant.
In a specific embodiment of the invention, the enzymatic hydrolysate after the secondary biological enzymolysis is centrifuged for 10-30min at 6000-8000 r/min and 2-4 ℃ in a centrifuge, and the supernatant is collected; preferably, centrifugation is carried out at 7000r/min for 20 min.
In a specific embodiment of the present invention, the extraction process comprises the steps of: (8) filtering; and (9) filling.
In a specific embodiment of the present invention, the step (8) of filtering comprises: the supernatant collected above was filtered through a 0.1 μm filter under aseptic conditions.
In a specific embodiment of the present invention, the filling of step (9) comprises: and filling the filtrate to obtain the finished product.
Table 1 shows reagents and instruments for preparing collagen peptide from fresh skin of animals.
TABLE 1 reagent and apparatus for preparing collagen peptide from fresh skin of animals
| Reagent/instrument | Model/specification | Manufacturer of the product |
| Bone breaking machine | RL-22S | Wuhan Sharp food machinery Co Ltd |
| Ultrapure water machine | UPR-11-40L | SICHUAN ULUPURE ULTRAPURE TECHNOLOGY Co.,Ltd. |
| Ultrasonic extractor | Scientz-50T | NINGBO SCIENTZ BIOTECHNOLOGY Co.,Ltd. |
| High-speed refrigerated centrifuge | TGC-16M | HUNAN MICHAEL EXPERIMENTAL APPARATUS Co.,Ltd. |
| High-pressure homogenizer | ATS-AH08-100 | ANTOS NANO TECHNOLOGY (SUZHOU) Co.,Ltd. |
| Pepsin | 9001-75-6 | solarbio |
| Trypsin | T8151 | solarbio |
Example 1 preparation of collagen peptide derived from donkey skin
1. Pretreating fresh skin: washing 5kg fresh donkey skin with purified water for 3 times, and removing surface impurities, fascia, and adipose tissue. Cleaning with purified water for 2 times, removing excessive water, mincing with meat mincer, weighing 4.8kg, and collecting minced donkey skin tissue, i.e. pretreated skin material.
2. Ultrasonic extraction: adding 3 times (W: V) of 14.4L of purified water into the minced donkey skin tissue, extracting at low temperature for 30min in an ultrasonic extractor, wherein the temperature is 2-4 ℃, the ultrasonic power is 400W/L, and the stirring speed is 1000r/min, so as to obtain tissue fluid after ultrasonic extraction.
3. High-pressure homogenization: and (3) homogenizing the tissue fluid subjected to ultrasonic extraction in a high-pressure homogenizer at a low temperature, wherein the homogenization is carried out at the temperature of 0-10 ℃, the homogenization pressure of 5000-7000Pa and the homogenization speed of 500ml/min, and the homogenization is carried out repeatedly for 3 times to obtain the homogenized tissue fluid subjected to high-pressure homogenization.
4. Extracting and separating the glue juice: steaming the homogenized tissue fluid in a high-pressure steaming kettle at 121 ℃ and 0.2MPa for 1.5 h; filtering the glue juice and the skin tissue by a filtering screen of 80 meshes, 120 meshes, 200 meshes, 300 meshes, 400 meshes and 500 meshes step by step, and separating to obtain 14.5L filtrate and separated skin tissue.
Adding 7.2L (4.8 × 1.5 ═ 7.2) purified water 1.5 times the mass-volume ratio of the pretreated skin tissue obtained in step 1, and performing gel extraction and separation again according to the step 2-4; repeatedly extracting and separating for 3 times; separating to obtain filtrate and separated skin tissue. Mixing the filtrates to obtain 29.0L of the extract. 289g of donkey hide tissue residue are obtained.
5. Primary concentration and foam extraction: and putting the collected glue juice extracting solution into an open pot, and heating for primary concentration. Stirring continuously during primary concentration, evaporating water, removing floating foam (namely foam extraction) generated in the concentration process, slightly extracting foam along the pot edge by using a wooden spoon during foam extraction, adding a proper amount of purified water, and repeating the foam extraction operation until the glue juice is transparent and no floating foam is generated, wherein about 7.8L of the glue juice after primary concentration and foam extraction is finished is obtained after primary concentration and foam extraction are finished.
6. Second-stage bionic enzymolysis: and (3) performing secondary bionic enzymolysis on the glue juice subjected to primary concentration and foam extraction, namely simulating the environments of artificial gastric juice and intestinal juice, and performing enzymolysis on the glue juice. The method comprises the following two steps:
I. first-order biological enzymolysis (simulated artificial gastric juice): adjusting the pH value of the gel juice after the primary concentration and foam extraction to 2.0 by using 2.0mol/L HCl and 2mol/L NaOH, adding 62.4g of pepsin (10 ten thousand U/g) according to the amount of 0.8 percent of the material percentage, namely the volume percentage of the gel juice, and carrying out enzymolysis for 2.0-2.5 h at the temperature of 37 +/-0.5 ℃. Heating to 90-100 ℃, and inactivating enzyme for 15min to obtain enzymatic hydrolysate after primary biological enzymolysis.
Secondary biological enzymatic hydrolysis (simulated human factory intestinal fluid): regulating the pH value of the enzymatic hydrolysate after the first-stage biological enzymolysis to 8.0 by using a phosphate buffer solution, adding 39.0g of pancreatin (4000U/g) according to the amount of 0.5 percent of the material percentage, namely the volume percentage of the glue juice liquid, and carrying out enzymolysis for 2.0-2.5 h at 50 +/-0.5 ℃. Heating to 90-100 ℃, and inactivating enzyme for 15min to obtain enzymatic hydrolysate after secondary biological enzymolysis.
7. Centrifuging and collecting enzymolysis liquid: and (3) centrifuging the enzymatic hydrolysate subjected to secondary biological enzymolysis for 20min in a high-capacity low-temperature centrifuge at the temperature of 2-4 ℃ under the 7000r/min, collecting supernate, and storing the supernate in a liquid storage tank.
8. And (3) filtering: the supernatant collected above was filtered through a 0.1 μm filter under aseptic conditions to obtain 7.5L of filtrate.
9. Filling: and (3) quantitatively filling 10ml of the filtrate to obtain a finished product, wherein the finished product is yellowish or yellow clear liquid.
Example 2 preparation of bovine-derived collagen peptide
1. Pretreating fresh skin: 5kg of dittany bark was washed 3 times with purified water to remove surface impurities, fascia, and adipose tissue. Cleaning with purified water for 2 times, removing excessive water, mincing with meat mincer, weighing 4.5kg, and collecting minced cowhide tissue, i.e. pretreated skin material.
2. Ultrasonic extraction: adding 13.5L purified water (W: V) in an amount of 17.8L) which is 3 times that of the minced cowhide tissue, and extracting at low temperature for 30min in an ultrasonic extractor, wherein the temperature is 2-4 ℃, the ultrasonic power is 400W/L, and the stirring speed is 1000r/min to obtain tissue fluid after ultrasonic extraction.
3. High-pressure homogenization: and (3) homogenizing the tissue fluid subjected to ultrasonic extraction in a high-pressure homogenizer at a low temperature, wherein the homogenization is carried out at the temperature of 0-10 ℃, the homogenization pressure of 5000-7000Pa and the homogenization speed of 500ml/min, and the homogenization is carried out repeatedly for 3 times to obtain the homogenized tissue fluid subjected to high-pressure homogenization.
4. Extracting and separating the glue juice: steaming the homogenized tissue fluid in a high-pressure steaming kettle at 121 ℃ and 0.2MPa for 1.5 h; filtering the glue juice and the skin tissue by a filtering screen of 80 meshes, 120 meshes, 200 meshes, 300 meshes, 400 meshes and 500 meshes step by step, and separating to obtain 13.8L of filtrate and separated skin tissue.
Adding 1.5 times of 6.75L of purified water into the obtained separated skin tissue according to the mass-volume ratio of the pretreated skin material in the step 1, and extracting and separating the glue juice according to the step 2-4; repeatedly extracting and separating for 3 times; separating to obtain filtrate and separated skin tissue. Mixing the filtrates to obtain 27.2L of the extract. 264g of cow leather tissue residue was obtained.
5. Primary concentration and foam extraction: and putting the collected glue juice extracting solution into an open pot, and heating for primary concentration. Stirring continuously during primary concentration, evaporating water, removing floating foam (namely foam extraction) generated in the concentration process, slightly extracting foam along the pot edge by using a wooden spoon during foam extraction, adding a proper amount of purified water, and repeating the foam extraction operation until the glue juice is transparent and no floating foam is generated, wherein about 7.2L of the glue juice after primary concentration and foam extraction is finished is obtained after primary concentration and foam extraction are finished.
6. Second-stage bionic enzymolysis: and (3) performing secondary bionic enzymolysis on the glue juice subjected to primary concentration and foam extraction, namely simulating the environments of artificial gastric juice and intestinal juice, and performing enzymolysis on the glue juice. The method comprises the following two steps:
I. first-order biological enzymolysis (simulated artificial gastric juice): adjusting the pH value of the gel juice after the initial concentration and foam extraction to 2.0 by using 2.0mol/L HCl and 2mol/L NaOH, adding 54.4g of pepsin (10 ten thousand U/g) according to the amount of 0.8 percent of the material percentage, namely the volume percentage of the gel juice, and carrying out enzymolysis for 2.0-2.5 h at the temperature of 37 +/-0.5 ℃. Heating to 90-100 ℃, and inactivating enzyme for 15min to obtain enzymatic hydrolysate after primary biological enzymolysis.
Secondary biological enzymatic hydrolysis (simulated human factory intestinal fluid): the pH value of the enzymatic hydrolysate after the first-stage biological enzymolysis is adjusted to 8.0 by using a phosphate buffer solution, 34.0g of pancreatin (4000U/g) is added according to the amount of 0.5 percent of the material percentage, namely the volume percentage of the glue juice, and the enzymatic hydrolysis is carried out for 2.0h to 2.5h at 50 +/-0.5 ℃. Heating to 90-100 ℃, and inactivating enzyme for 15min to obtain enzymatic hydrolysate after secondary biological enzymolysis.
7. Centrifuging and collecting enzymolysis liquid: and (3) centrifuging the enzymatic hydrolysate subjected to secondary biological enzymolysis for 20min in a high-capacity low-temperature centrifuge at the temperature of 2-4 ℃ under the 7000r/min, collecting supernate, and storing the supernate in a liquid storage tank.
8. And (3) filtering: the supernatant collected above was filtered through a 0.1 μm filter under aseptic conditions to obtain 7.0L of filtrate.
9. Filling: and (3) quantitatively filling 10ml of the filtrate to obtain a finished product, wherein the finished product is yellowish or yellow clear liquid.
Example 3 preparation of porcine skin-derived collagen peptide
1. Pretreating fresh skin: the fresh pigskin of 5kg is treated and washed 3 times with purified water to remove surface impurities, fascia, and adipose tissue. Cleaning with purified water for 2 times, removing excessive water, mincing with meat mincer, weighing 2.8kg, and collecting minced Corii Sus Domestica tissue, i.e. pretreated skin material.
2. Ultrasonic extraction: adding 3 times (W: V) of 8.4L of purified water into the minced pigskin tissue, wherein the total volume is 11.0L, and extracting for 30min at low temperature in an ultrasonic extractor, wherein the temperature is 2-4 ℃, the ultrasonic power is 400W/L, and the stirring speed is 1000r/min, so as to obtain tissue fluid after ultrasonic extraction.
3. High-pressure homogenization: and (3) homogenizing the tissue fluid subjected to ultrasonic extraction in a high-pressure homogenizer at a low temperature, wherein the homogenization is carried out at the temperature of 0-10 ℃, the homogenization pressure of 5000-7000Pa and the homogenization speed of 500ml/min, and the homogenization is carried out repeatedly for 3 times to obtain the homogenized tissue fluid subjected to high-pressure homogenization.
4. Extracting and separating the glue juice: steaming the homogenized tissue fluid in a high-pressure steaming kettle at 121 ℃ and 0.2MPa for 1.5 h; filtering the glue juice and the skin tissue by a filtering screen of 80 meshes, 120 meshes, 200 meshes, 300 meshes, 400 meshes and 500 meshes step by step, and separating to obtain 10.8L of filtrate and separated skin tissue.
Adding 1.5 times of 4.2L of purified water into the obtained separated skin tissue according to the mass-volume ratio of the pretreated skin material in the step 1, and performing gel extraction and separation according to the step 2-4; repeatedly extracting and separating for 3 times; separating to obtain filtrate and separated skin tissue.
Mixing the filtrates to obtain 18.9L of succus mucilage extract. 76.3g of pigskin tissue residue was obtained.
5. Primary concentration and foam extraction: and putting the collected glue juice extracting solution into an open pot, and heating for primary concentration. Stirring continuously during primary concentration, evaporating water, removing floating foam (namely foam extraction) generated in the concentration process, slightly extracting foam along the pot edge by using a wooden spoon during foam extraction, adding a proper amount of purified water, and repeating the foam extraction operation until the glue juice is transparent and no floating foam is generated, wherein about 4.9L of the glue juice after primary concentration and foam extraction is finished is obtained after primary concentration and foam extraction are finished.
6. Second-stage bionic enzymolysis: and (3) performing secondary bionic enzymolysis on the glue juice subjected to primary concentration and foam extraction, namely simulating the environments of artificial gastric juice and intestinal juice, and performing enzymolysis on the glue juice. The method comprises the following two steps:
I. first-order biological enzymolysis (simulated artificial gastric juice): adjusting the pH value of the gel juice after the primary concentration and foam extraction to 2.0 by using 2.0mol/L HCl and 2mol/L NaOH, adding 39.2g of pepsin (10 ten thousand U/g) according to the amount of 0.8 percent of the material percentage, namely the volume percentage of the gel juice, and carrying out enzymolysis for 2.0-2.5 h at the temperature of 37 +/-0.5 ℃. Heating to 90-100 ℃, and inactivating enzyme for 15min to obtain enzymatic hydrolysate after primary biological enzymolysis.
Secondary biological enzymatic hydrolysis (simulated human factory intestinal fluid): the pH value of the enzymatic hydrolysate after the first-stage biological enzymolysis is adjusted to 8.0 by using a phosphate buffer solution, 24.5g of pancreatin (4000U/g) is added according to the amount of 0.5 percent of the material percentage, namely the volume percentage of the glue juice, and the enzymatic hydrolysis is carried out for 2.0h to 2.5h at 50 +/-0.5 ℃. Heating to 90-100 ℃, and inactivating enzyme for 15min to obtain enzymatic hydrolysate after secondary biological enzymolysis.
7. Centrifuging and collecting enzymolysis liquid: and (3) centrifuging the enzymatic hydrolysate subjected to secondary biological enzymolysis for 20min in a high-capacity low-temperature centrifuge at the temperature of 2-4 ℃ under the 7000r/min, collecting supernate, and storing the supernate in a liquid storage tank.
8. And (3) filtering: the supernatant collected above was filtered through a 0.1 μm filter under aseptic conditions to obtain 4.7L of filtrate.
9. Filling: and (3) quantitatively filling 10ml of the filtrate to obtain a finished product, wherein the finished product is yellowish or yellow clear liquid.
Example 4 preparation of equine dermatic collagen peptide
1. Pretreating fresh skin: the 5kg of cortex Dictamni was treated with purified water and washed 3 times to remove surface impurities, fascia, and adipose tissue. Cleaning with purified water for 2 times, removing excessive water, mincing with meat mincer, weighing 4.9kg, and collecting minced horse skin tissue, i.e. pretreated skin material.
2. Ultrasonic extraction: adding 14.7L purified water (W: V) which is 3 times of the amount of the minced horse skin tissue, and extracting at low temperature for 30min in an ultrasonic extractor, wherein the temperature is 2-4 ℃, the ultrasonic power is 400W/L, and the stirring speed is 1000r/min to obtain tissue fluid after ultrasonic extraction.
3. High-pressure homogenization: and (3) homogenizing the tissue fluid subjected to ultrasonic extraction in a high-pressure homogenizer at a low temperature, wherein the homogenization is carried out at the temperature of 0-10 ℃, the homogenization pressure of 5000-7000Pa and the homogenization speed of 500ml/min, and the homogenization is carried out repeatedly for 3 times to obtain the homogenized tissue fluid subjected to high-pressure homogenization.
4. Extracting and separating the glue juice: steaming the homogenized tissue fluid in a high-pressure steaming kettle at 121 ℃ and 0.2MPa for 1.5 h; filtering the glue juice and the skin tissue by a filtering screen of 80 meshes, 120 meshes, 200 meshes, 300 meshes, 400 meshes and 500 meshes step by step, and separating to obtain 19.6L of filtrate and separated skin tissue.
Adding 1.5 times of 7.35L of purified water into the obtained separated skin tissue according to the mass-volume ratio of the pretreated skin material in the step 1, and performing glue extraction and separation again according to the step 2-4; repeatedly extracting and separating for 3 times; separating to obtain filtrate and separated skin tissue. Mixing the filtrates to obtain 34.0L of succus mucilage extract. 318g of horse skin tissue residues are obtained.
5. Primary concentration and foam extraction: and putting the collected glue juice extracting solution into an open pot, and heating for primary concentration. Stirring continuously during primary concentration, evaporating water, removing floating foam (namely foam extraction) generated in the concentration process, slightly extracting foam along the pot edge by using a wooden spoon during foam extraction, adding a proper amount of purified water, and repeating the foam extraction operation until the glue juice is transparent and no floating foam is generated, wherein about 9.2L of the glue juice after primary concentration and foam extraction is finished is obtained after primary concentration and foam extraction are finished.
6. Second-stage bionic enzymolysis: and (3) performing secondary bionic enzymolysis on the glue juice subjected to primary concentration and foam extraction, namely simulating the environments of artificial gastric juice and intestinal juice, and performing enzymolysis on the glue juice. The method comprises the following two steps:
I. first-order biological enzymolysis (simulated artificial gastric juice): adjusting the pH value of the gel juice after the primary concentration and foam extraction to 2.0 by using 2.0mol/L HCl and 2mol/L NaOH, adding 73.6g of pepsin (10 ten thousand U/g) according to the amount of 0.8 percent of the material percentage, namely the volume percentage of the gel juice, and carrying out enzymolysis for 2.0-2.5 h at the temperature of 37 +/-0.5 ℃. Heating to 90-100 ℃, and inactivating enzyme for 15min to obtain enzymatic hydrolysate after primary biological enzymolysis.
Secondary biological enzymatic hydrolysis (simulated human factory intestinal fluid): regulating the pH value of the enzymatic hydrolysate after the first-stage biological enzymolysis to 8.0 by using a phosphate buffer solution, adding 46.0g of pancreatin (4000U/g) according to the amount of 0.5 percent of the material percentage, namely the volume percentage of the glue juice liquid, and carrying out enzymolysis for 2.0 to 2.5 hours at 50 +/-0.5 ℃. Heating to 90-100 ℃, and inactivating enzyme for 15min to obtain enzymatic hydrolysate after secondary biological enzymolysis.
7. Centrifuging and collecting enzymolysis liquid: and (3) centrifuging the enzymatic hydrolysate subjected to secondary biological enzymolysis for 20min in a high-capacity low-temperature centrifuge at the temperature of 2-4 ℃ under the 7000r/min, collecting supernate, and storing the supernate in a liquid storage tank.
8. And (3) filtering: the supernatant collected above was filtered through a 0.1 μm filter under aseptic conditions to obtain 9.0L of filtrate.
9. Filling: and (3) quantitatively filling 10ml of the filtrate to obtain a finished product, wherein the finished product is yellowish or yellow clear liquid.
Example 5 measurement of polypeptide content in different solutions of collagen peptides derived from skin
The polypeptide content of the different collagen peptide solutions of skin origin prepared in examples 1 to 4 was determined by the Folin phenol method. Table 2 shows the collagen peptide content detection reagent and apparatus.
TABLE 2 collagen peptide content detection reagent and instrument
The specific operation steps are as follows:
I. preparation of control solutions
Bovine serum albumin control (batch No. PB10056)0.30g was weighed precisely, and water was added to 100ml to prepare a solution containing bovine serum albumin 0.03mg per 1.0 ml.
Preparation of sample to be examined
The donkey-hide-derived collagen peptide solution, the cow-hide-derived collagen peptide solution, the pig-hide-derived collagen peptide solution and the horse-hide-derived collagen peptide solution prepared in the embodiments 1 to 4 were respectively sampled by 1.0ml, and water was added to 200ml to be used as samples to be detected. Each sample was done in 3 replicates.
Preparation of the Standard Curve
Precisely measuring 0.0 ml, 0.1 ml, 0.3 ml, 0.5 ml, 0.7 ml and 0.9ml of the reference substance solution prepared in the step I, respectively placing the reference substance solution in test tubes with scales, adding water to 1ml, respectively adding 1ml of alkaline copper solution (mixed solution of copper sulfate pentahydrate, potassium tartrate and sodium hydroxide), shaking uniformly, adding 4ml of the test solution of Folin phenol, immediately mixing uniformly, placing in a water bath at 55 ℃ for 10 minutes, and measuring the absorbance at the wavelength of 650 nm; meanwhile, a No. 0 tube is used as a blank control, the absorbance is used as a vertical coordinate, and the concentration of the control solution is used as a horizontal coordinate to draw a standard curve.
And IV, precisely measuring 1.0ml of the collagen peptide sample to be detected prepared in the step II, determining the concentration of the sample solution to be detected from the standard curve according to a method under the preparation item of the standard curve in the step III from the addition of the alkaline copper test solution, and multiplying the concentration by the dilution times to obtain the polypeptide content of the collagen peptide solution prepared in the embodiments 1-4 (Table 3).
TABLE 3 detection results of different collagen peptide derived from skin
Example 6 determination of the content of L-hydroxyproline, glycine, alanine, L-proline in different collagen peptides derived from skin
Referring to the part of the 'Chinese pharmacopoeia' 2015 edition, the content of L-hydroxyproline, glycine, alanine and L-proline in the collagen peptide solution of different skin sources prepared in the embodiments 1 to 4 is detected by a method for measuring the content of L-hydroxyproline, glycine, alanine and L-proline in the donkey-hide gelatin by applying high performance liquid chromatography under the item of donkey-hide gelatin. Therefore, the content difference of L-hydroxyproline, glycine, alanine and L-proline in the collagen peptide prepared by the traditional donkey-hide gelatin and the method is compared. Table 4 shows the content of amino acids in different collagen peptides from the skin and the instrument.
TABLE 4 detection reagent and instrument for amino acid content in different collagen peptides derived from skin
| Reagent/instrument | Model/specification | Manufacturer of the product |
| Acetonitrile | M157-4 | MREDA |
| Sodium acetate | 500G/bottle, super grade pure | TIANJIN DAMAO CHEMICAL REAGENT FACTORY |
| Acetic acid | 500 ml/bottle, super grade pure | TIANJIN FUYU FINE CHEMICAL Co.,Ltd. |
| Phenyl Isothiocyanate (PITC) | 103-72-0 | Ron's reagent |
| Triethylamine | 121-44-8 | Ron's reagent |
| L-hydroxyproline reference substance | 1115-201602 | NATIONAL INSTITUTES FOR FOOD AND DRUG CONTROL |
| Glycine reference | 140689-201605 | NATIONAL INSTITUTES FOR FOOD AND DRUG CONTROL |
| Alanine control | 140680-201604 | NATIONAL INSTITUTES FOR FOOD AND DRUG CONTROL |
| L-proline reference | 111659-200301 | NATIONAL INSTITUTES FOR FOOD AND DRUG CONTROL |
| High performance liquid chromatograph | S3000 | Hua spectral New science and technology Co Ltd |
The specific operation method comprises the following steps: referring to the part of Chinese pharmacopoeia 2015 edition, the content of L-hydroxyproline, glycine, alanine and L-proline in different collagen peptides derived from skin is determined by a method for determining the content of donkey-hide gelatin under item, namely high performance liquid chromatography.
The method comprises the following steps:
I. chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; gradient elution is carried out by taking acetonitrile-0.1 mol/L sodium acetate solution (pH value is adjusted to 6.5 by acetic acid) as a mobile phase A and acetonitrile-water (4:1) as a mobile phase B according to the specification in the following table; the detection wavelength is 254 nm; the column temperature was 43 ℃. The theoretical plate number is not lower than 4000 calculated according to the peak of L-hydroxyproline.
TABLE 5 chromatographic conditions
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0～11 | 100→93 | 0→7 |
| 11～13.9 | 93→88 | 7→12 |
| 13.9～14 | 88→85 | 12→15 |
| 14～29 | 85→66 | 15→34 |
| 29～30 | 66→0 | 34→100 |
Preparation of control solutions: taking appropriate amount of L-hydroxyproline reference substance, glycine reference substance, alanine reference substance, and L-proline reference substance, precisely weighing, and adding 0.1mol/L hydrochloric acid solution to obtain 1ml mixed solution containing L-hydroxyproline 80 μ g, glycine 0.16mg, alanine 70 μ g, and L-proline 0.12 mg.
Preparation of test solution: after freeze-drying the collagen peptide liquid derived from different animal skins obtained in the examples 1-4, respectively taking 0.25g, precisely weighing, respectively placing in a 25ml measuring flask, adding 20ml of 0.1mol/L hydrochloric acid solution, performing ultrasonic treatment (500W, 40kHz) for 30min, cooling, adding 0.1mol/L hydrochloric acid solution to a constant volume to a scale, and shaking up. Precisely weighing 2ml, placing the weighed 2ml in a 5ml ampoule, adding 2ml hydrochloric acid, hydrolyzing at 150 ℃ for 1h, cooling, transferring to an evaporation dish, washing with 10ml water for several times, merging the washing solution into the evaporation dish, evaporating to dryness, dissolving the residue in 0.1mol/L hydrochloric acid solution, transferring to a 25ml measuring flask, adding 0.1mol/L hydrochloric acid solution to the scale, and shaking up to obtain the product.
Precisely weighing 5ml of each of the reference solution and the sample solution, respectively placing the reference solution and the sample solution into 25ml measuring bottles, adding 2.5ml of acetonitrile solution of 0.1mol/L phenyl isothiocyanate and 2.5ml of acetonitrile solution of 1mol/L triethylamine, shaking uniformly, placing the bottles at room temperature for 1h, adding 50% acetonitrile to the scale, and shaking uniformly. Adding n-hexane 10ml into 10ml, shaking, standing for 10min, collecting the lower layer solution, and filtering to obtain the filtrate.
Assay method: and (3) respectively filling 1ml of the reference substance solution and the test substance solution subjected to derivatization in the step III into a special sample bottle, placing the special sample bottle in a sample disc of a liquid chromatograph, automatically loading the sample for detection (the loading amount is 5 mu l), and recording a chromatogram.
V, detection result: the content of L-hydroxyproline, glycine, alanine and L-proline in the collagen peptides obtained from different animal skin sources in examples 1-4 was measured by an internal standard method. The results are shown in FIGS. 2-6 and Table 6. The results show that the content of L-hydroxyproline, glycine, alanine and L-proline in the collagen peptides obtained from different animal skin sources in examples 1-4 is far higher than that of donkey-hide gelatin in Chinese pharmacopoeia. Therefore, the method can effectively extract the collagen peptides from different animal skin sources, takes four amino acids of L-hydroxyproline, glycine, alanine and L-proline as markers, and has the extraction rate far higher than that of the donkey-hide gelatin prepared by the traditional method compared with the donkey-hide gelatin.
TABLE 6 detection results of L-hydroxyproline, glycine, alanine, and L-proline contents in different collagen peptides derived from skin sources
Example 7
The molecular weight range of small molecular peptides in the collagen peptide solution derived from donkey skin of example 1 was determined by SDS-PAGE and compared with marine fish collagen peptide powder (Tongrentang, lot No. 8204001) and donkey-hide gelatin polypeptide powder (Xian Wang breed science and technology Biotech Co., Ltd.) purchased. Table 7 shows the molecular weight detection reagents and apparatus for collagen peptide.
TABLE 7 collagen peptide molecular weight detection reagent and instrument
The specific experimental steps are as follows:
1. glue making
TABLE 8 Gum-making reagents and gum concentrations
The glue volume ratio of 3 glues is about: 4:1.5:1, preparing the separation glue and the interlayer glue, carefully injecting purified water to seal the glue, pouring out the covering water layer after the glue is completely polymerized, and then sucking residual water by using filter paper.
2. Sample treatment: after the sample was concentrated, 1ml of the loading buffer was added and boiled in hot water for 5 minutes.
3. Sample application: the marker (marker) was spotted into the sample at 15. mu.l each, at about 15. mu.g protein peptide content.
4. Glue running: the first three hours voltage 50(20-30mA), the second two hours 90(30 mA).
5. Fixing: adding the glue into the fixing solution, fixing for 20 minutes, and repeatedly cleaning with purified water.
6. Dyeing: staining solution was added for overnight staining.
7. And (3) decoloring: discarding the staining solution, adding a decolorizing solution, decolorizing for 40 min on a shaking table (60 revolutions), and repeatedly cleaning with purified water.
8. Protein bands were visualized.
9. Preparing a reagent:
9.1 acrylamide: acrylamide 48g, N, N-methylene bisacrylamide 1.5g, and water is added to the mixture to reach the constant volume of 100ml, so that the traditional Chinese medicine is obtained.
9.2 gel buffer: 0.3g of SDS and 36.4g of Tris alkali (Tris-base), adding water to a constant volume of 100ml, and adjusting the pH value to 8.45 by hydrochloric acid to obtain the finished product.
9.3 Anode buffer: 12.11g of Tris alkali (Tris-base), adding water to a constant volume of 500ml, and adjusting the pH value to 8.9 by hydrochloric acid to obtain the finished product.
9.4 cathode buffer: 6.06g of Tris alkali (Tris-base), 8.96g of Tricine and 0.5g of SDS, and adding water to a constant volume of 500ml to obtain the finished product.
9.5 Loading buffer: SDS 0.8g, glycerin 4g, beta-mercaptoethanol 0.2g, bromophenol blue 0.01g, add 0.5mol/LTris-HCl pH6.8 to dissolve to 10ml, get final product.
9.6 stationary liquid: 50ml of ethanol, 10ml of glacial acetic acid and 40ml of water are mixed evenly to obtain the product.
9.7 staining solution: 4ml of phosphoric acid, 20g of ammonium sulfate, 2500.05g of Coomassie brilliant blue and 50ml of ethanol, and adding water to a constant volume of 250ml to obtain the compound.
9.8 decolorizing solution: 45ml of ethanol, 10ml of glacial acetic acid and 45ml of water are mixed evenly to obtain the product.
9.910% APS: 1g of ammonium persulfate is dissolved in 10ml of water.
10. As a result:
as shown in the SDS-PAGE results of the small-molecule polypeptide in FIG. 6, the molecular weight of the donkey-hide collagen peptide (lane 3) obtained in example 1 of the present invention is about 2000Da and 5000Da, there are 2 distinct bands, the molecular weight of the marine fish collagen peptide powder (lane 1) is 3 concentrated bands between 4600-10000Da, the molecular weight of the purchased donkey-hide gelatin polypeptide (lane 2) is 1 distinct band at 4600Da, and some bands slightly below 10000Da have 1 shallower peak.
Therefore, the molecular weight range of the animal skin-derived collagen peptide obtained by the method is equivalent to the molecular weight of purchased donkey-hide gelatin polypeptide powder; compared with the molecular weight of the marine fish collagen peptide powder, the animal skin collagen peptide obtained by the invention has smaller molecular weight and is easier to absorb by organisms. The animal skin collagen body obtained by the invention is small molecular peptide. The donkey-hide gelatin has high viscosity, the main component of the donkey-hide gelatin is collagen macromolecules, and the modern traditional Chinese medicine pharmacological research proves that the donkey-hide gelatin plays a role of micromolecular peptide, so that the donkey-hide gelatin can cause dyspepsia, abdominal distension and poor digestion and absorption when being taken for the elderly and the weak spleen and stomach. The animal skin collagen peptide, especially donkey skin collagen peptide, has the effects of nourishing blood and enriching blood, improving immunity and the like of donkey-hide gelatin, and avoids adverse reaction of the donkey-hide gelatin due to small molecular weight and easy absorption.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. An extraction process of animal skin collagen peptide, wherein the extraction process comprises the following steps: ultrasonic extraction, high-pressure homogenization and secondary bionic enzymolysis.
2. The process for extracting animal skin collagen peptide according to claim 1, wherein said process further comprises a step selected from one or more of: pretreating animal skin, extracting and separating glue juice, primarily concentrating and foam extracting, collecting enzymolysis liquid, filtering and filling;
preferably, the animal skin is animal hide;
preferably, the animal skin is selected from donkey hide, horse hide, cow hide or pig hide.
3. The process for extracting animal skin collagen peptide according to claim 1 or 2, wherein the extraction process comprises the steps of:
(1) pretreating animal skins;
(2) ultrasonic extraction;
(3) homogenizing under high pressure;
(4) extracting and separating the glue juice;
(5) primary concentration and foam extraction;
(6) performing secondary bionic enzymolysis;
(7) and collecting the enzymolysis liquid.
4. The process for extracting collagen peptide from animal skin according to any one of claims 1 to 3, wherein the step (1) of pretreating animal skin comprises: mincing animal skin to obtain minced animal skin tissue, namely pretreated skin material;
preferably, skin surface impurities, fascia and fat tissue are removed, and then the animal skin is minced to obtain minced animal skin tissue;
preferably, the animal skin treatment is washed for a plurality of times by using water, preferably purified water, preferably for 2-3 times, then impurities, fascia and fat tissues on the surface of the skin are removed, and then the animal skin is minced to obtain minced animal skin tissues.
5. The process for extracting animal skin collagen peptide according to any one of claims 1 to 4, wherein the ultrasonic extraction of step (2) comprises: adding water with multiple volumes into the minced animal skin tissue, and extracting in an ultrasonic extractor to obtain tissue fluid after ultrasonic extraction;
preferably, 3-5 times of water is added into the minced animal skin tissues, and more preferably, 3 times of water is added;
preferably, low-temperature extraction is carried out in an ultrasonic extractor, and more preferably, the extraction temperature is 2-4 ℃;
preferably, the ultrasonic power is 300-500 w/L;
preferably, the stirring speed is 1000-1200 r/min;
preferably, the ultrasonic time is 30-50 min;
more preferably, the ultrasonic power is 400w/L, the stirring speed is 1000r/min, and the ultrasonic time is 30 min.
6. The process for extracting collagen peptide according to any one of claims 1 to 5, wherein the step (3) of high pressure homogenization comprises: carrying out high-pressure homogenization on the tissue fluid obtained in the step (2) after ultrasonic extraction in a high-pressure homogenizer to obtain homogenized tissue fluid after high-pressure homogenization;
preferably, the high pressure homogenization is performed at low temperature; preferably, the high-pressure homogenization temperature is 0-10 ℃;
preferably, the high-pressure homogenization is repeated for multiple times, preferably 3-5 times, preferably 3 times;
preferably, the homogenizing shear is carried out at a homogenizing pressure of 5000-7000Pa, preferably 6000 Pa;
preferably, the homogenizing shear is carried out at a homogenizing rate of 300-700ml/min, preferably 500 ml/min.
7. The process for extracting collagen peptide according to any one of claims 1 to 6, wherein the step (4) of extracting and separating the gel juice comprises: cooking the homogenized tissue fluid obtained in the step (3) in a cooking kettle to obtain glue juice and a skin tissue; separating the glue juice from the skin tissue by filtration to obtain filtrate and separated skin tissue, and collecting filtrate to obtain glue juice extract;
preferably, the mixture is cooked in a high-pressure cooking kettle, preferably at 121 ℃ and 0.2MPa for 1.5 to 2 hours, preferably for 1.5 hours;
preferably, the glue juice and the skin tissue are separated by a step-by-step filtration method, preferably a filtration screen with 80 meshes, 120 meshes, 200 meshes, 300 meshes, 400 meshes and 500 meshes, and the glue juice extract and the separated skin tissue are collected;
preferably, adding water with multiple volumes, preferably 1-2 times water, more preferably 1.5 times water into the separated skin tissue according to the mass-volume ratio of the pretreated skin tissue in the step (1), repeatedly performing gel extraction and separation for multiple times to obtain filtrate and the separated skin tissue, combining the filtrate, and collecting the filtrate to obtain a gel extract; preferably, the extraction and separation of the glue juice are repeated for 2-5 times, more preferably 3 times.
8. The process for extracting collagen peptide according to any one of claims 1 to 7, wherein the step (5) of primary concentration and foam extraction comprises: heating and primarily concentrating the glue juice extracting solution collected in the step (4); removing froth generated in the concentration process, namely foam extraction; obtaining the glue juice after the primary concentration and foam extraction;
preferably, stirring is continuously carried out during the primary concentration;
preferably, when the foam is extracted, a wooden spoon is used for slightly extracting the foam along the pot edge, and a proper amount of water, preferably purified water, is added;
preferably, the foam extraction operation is repeated continuously until the glue juice is transparent and no froth is generated, and the glue juice after the initial concentration and the foam extraction is obtained after the initial concentration and the foam extraction are finished.
9. The process for extracting collagen peptide from animal skin according to any one of claims 1 to 8, wherein the secondary biomimetic enzymatic hydrolysis in step (6) comprises: and (3) carrying out secondary bionic enzymolysis on the primary concentrated and foam-extracted glue juice obtained in the step (5) to carry out enzymolysis on the glue juice, wherein the secondary bionic enzymolysis comprises the following steps:
I. first-order biological enzymolysis (simulated artificial gastric juice):
adjusting the pH value of the glue juice after the primary concentration and foam extraction to 1.5-2.5 by using acid or alkali, preferably adjusting the pH value to 2.0, and adding pepsin (10 ten thousand U/g) into the glue juice after the primary concentration and foam extraction according to the volume percentage of 0.5-1.0% of the glue juice after the primary concentration and foam extraction to obtain an enzymatic hydrolysate after primary biological enzymolysis;
preferably, the acid is selected from one or more of hydrochloric acid, phosphoric acid, sulphuric acid, oxalic acid, acetic acid and citric acid, preferably HCl;
preferably, the base is selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate and sodium bicarbonate and triethanolamine, preferably NaOH;
more preferably, the pH is adjusted with 2.0mol/L HCl and/or 2mol/L NaOH;
preferably, 0.8% pepsin (10 ten thousand U/g) is added; preferably, enzymolysis is carried out for 2.0 h-2.5 h at 37 +/-0.5 ℃;
preferably, heating to 90-100 ℃ to inactivate the enzyme to obtain enzymatic hydrolysate after primary biological enzymolysis; preferably, the enzyme is inactivated for 15-20 min, and more preferably, the enzyme is inactivated for 15 min;
secondary biological enzymatic hydrolysis (simulated human factory intestinal fluid):
adjusting the pH value of the enzymatic hydrolysate after the primary biological enzymolysis to 7.5-8.5 by using a buffer solution, preferably adjusting the pH value to 8.0; adding pancreatin (4000U/g) into the pH-adjusted enzymatic hydrolysate after the primary biological enzymolysis according to the volume percentage of 0.5-1.0% of the glue juice after the primary concentration and foam extraction; obtaining enzymolysis liquid after the second-stage biological enzymolysis;
preferably, the buffer is a phosphate buffer;
preferably, 0.5% pancreatin (4000U/g) is added; more preferably, enzymolysis is carried out for 2.0 h-2.5 h at 50 ℃ +/-0.5 ℃;
preferably, heating to 90-100 ℃ to inactivate the enzyme to obtain enzymatic hydrolysate after secondary biological enzymolysis; preferably, the enzyme is inactivated for 15-20 min, more preferably, the enzyme is inactivated for 15 min.
10. The process for extracting an animal skin collagen peptide according to any one of claims 1 to 9, wherein said step (7) of collecting the enzymatic hydrolysate comprises centrifuging the enzymatic hydrolysate after the secondary biological enzymatic hydrolysis, and collecting the supernatant;
preferably, the enzymatic hydrolysate after the secondary biological enzymolysis is centrifuged for 10-30min in a centrifuge at the temperature of 2-4 ℃ and under the condition of 6000-8000 r/min, and supernate is collected; preferably, centrifugation is carried out for 20min under the condition of 7000 r/min;
preferably, the extraction process comprises the steps of: (8) filtering; and (9) filling;
preferably, the step (8) of filtering comprises: filtering the collected supernatant with 0.1 μm filter under aseptic condition;
preferably, the step (9) of filling comprises: and filling the filtrate to obtain the finished product.
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Application publication date: 20200818 |