CN108314726B - Giant salamander skin collagen extraction method and collagen product extracted by same - Google Patents
Giant salamander skin collagen extraction method and collagen product extracted by same Download PDFInfo
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 95
- 108010035532 Collagen Proteins 0.000 title claims abstract description 95
- 229920001436 collagen Polymers 0.000 title claims abstract description 95
- 241000157862 Dicamptodontidae Species 0.000 title claims abstract description 91
- 238000000605 extraction Methods 0.000 title abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 38
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000002253 acid Substances 0.000 claims abstract description 23
- 239000000049 pigment Substances 0.000 claims abstract description 8
- 238000004108 freeze drying Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 38
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 36
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 23
- 239000007864 aqueous solution Substances 0.000 claims description 22
- 238000002791 soaking Methods 0.000 claims description 22
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 18
- 239000011780 sodium chloride Substances 0.000 claims description 18
- 238000002386 leaching Methods 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 239000000047 product Substances 0.000 claims description 15
- 239000012266 salt solution Substances 0.000 claims description 13
- 238000000926 separation method Methods 0.000 claims description 11
- 235000013861 fat-free Nutrition 0.000 claims description 10
- 239000000287 crude extract Substances 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 8
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 8
- 239000003513 alkali Substances 0.000 claims description 8
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 7
- 235000004279 alanine Nutrition 0.000 claims description 7
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- 102000057297 Pepsin A Human genes 0.000 description 1
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Classifications
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- 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 or cold insoluble globulin [CIG]
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- Biochemistry (AREA)
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- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention relates to a giant salamander skin collagen extraction method and a collagen product extracted by the method. The method comprises the following steps: obtaining giant salamander skin, removing fat, removing pigment, extracting citric acid, performing primary precipitation, performing secondary precipitation, dialyzing, freeze-drying and the like. The amount of the collagen obtained by the acid extraction method is equal to or more than that of the collagen obtained by the enzyme extraction method, and the process is simpler. The collagen product extracted by the method of the invention contains comprehensive amino acid and has higher nutritive value.
Description
Technical Field
The invention relates to the technical field of extraction of animal collagen, in particular to a giant salamander skin collagen extraction method and a collagen product extracted by the method.
Background
Giant salamanders (Giant), also called Giant salamanders, belong to the order of the amphibia and the family of cryptogillidaceae, are the largest amphibians in the world, are the species of the transition species Giant salamanders between aquatic and terrestrial which are almost the same era as dinosaurs, have evolved very originally and are called "activated stones".
Giant salamanders are widely distributed in China, mainly inhabit branches in the middle and upper reaches of Yangtze river, yellow river and Zhujiang river, are unique rare wild animals in China, are listed as national secondary protection animals and cannot be killed, but artificially-bred second-generation giant salamanders can be used as edible aquatic products. China has started to artificially breed and domesticate giant salamanders, the number of the giant salamanders is greatly increased nowadays, and the artificially bred second-generation giant salamanders are allowed to be eaten. Due to the improvement of various protective measures and technologies, the giant salamander resource is in a rapid growth stage nowadays, and powerful guarantee is provided for the future development and utilization of the giant salamander resource.
Collagen (collagen) is an extracellular protein with three peptide chain structures, is widely distributed in a living body, has the highest content of collagen in all proteins in an animal body, reaches more than 80 percent of the content of crude protein, and is an important participant of the life activities of the animal body. In living bodies, collagen has a large specific gravity in content and is different in kind, and there are 28 kinds of collagen. The collagen has wide application and can be used as biomedical materials in medicine. Collagen has strong adhesion and smoothness to the skin, is used medically as a suture for wound healing, and has good smoothness so that it is not easily loosened at the knot of medical suture, so that the wound can be healed in a short time. Collagen drugs are also actively studied in the medical field, and most of these drugs are used for glaucoma treatment, ophthalmic anti-infection, and other purposes, as well as for local treatment of wounds and control of wound infection, gynecological cervical dysplasia, surgical local anesthesia, and the like. In tissue engineering, collagen and other extracellular substances constitute a tissue extracellular matrix, and thus is a natural scaffold material in the body of an organism. On food products, collagen can be used in the cooking of meat to increase meat firmness; can be used as a natural sausage casing product; and the collagen has the functions of immobilized enzyme and oxidation resistance, which has great improvement effect on the flavor and quality of meat products. In addition, collagen has a great number of applications in the fields of cosmetics, nutritional additives, and the like.
The basic principle of collagen extraction is to separate collagen from other proteins by changing the external environment of the proteins according to the characteristics of collagen. The extraction method can be divided into 4 types according to different extraction media: namely hot water extraction, acid extraction, alkaline extraction and enzymatic extraction. In addition, acid and alkali combined hot water extraction, enzyme combined extraction, etc. are also available.
When extracting collagen from giant salamander-derived materials, it is generally believed that the hot water method mostly denatures the obtained collagen into gelatin due to the higher extraction temperature; the alkaline process is rapid and complete, but all amino acids containing hydroxyl and sulfhydryl groups are destroyed and racemization (structural variation) occurs; the acid process yields are too low and the extraction times are long, and tryptophan is not stable to acids. Therefore, enzymatic or extraction methods including enzymatic steps are generally used to obtain high yields.
For example, CN201710501842.9 discloses a method for extracting collagen from giant salamander, which comprises the following steps: (1) sterilizing, stirring and pulping, acid leaching, water washing, alkaline leaching, secondary water washing, salt leaching, enzymolysis, purification and the like. The patent application discloses that even if an acid method is adopted in combination with one of alkaline leaching, salt leaching and enzymolysis, the extraction rate is about 40%.
For another example, CN201410630235.9 discloses a method for extracting collagen by utilizing ecdysis of giant salamander, which comprises the following steps: pretreating, performing enzymolysis and crude extraction, filtering, concentrating, spray drying and the like to obtain the collagen with the molecular weight of less than 3000 daltons.
However, enzymatic extraction also has disadvantages. For example, hydrolysis is not complete enough and may cause changes in the collagen structural component due to proteolytic cleavage to remove the non-helical telopeptides of collagen. In addition, extraction conditions such as pH are more demanding and expensive enzymes are used, and the cost of the extraction process is more expensive for large scale extraction.
The invention provides an optimized acid method for extracting collagen from giant salamander skin, and the method can obtain the collagen from the giant salamander skin with high extraction rate and full amino acid.
Disclosure of Invention
In order to solve the above problems in the art, the present invention provides in a first aspect a method for extracting collagen from giant salamander skin, wherein the method comprises the steps of:
(1) obtaining giant salamander skin; (2) soaking the giant salamander skin by using isopropanol to remove fat in the giant salamander skin to obtain fat-free giant salamander skin; (3) soaking the fat-free giant salamander skin by using alkali liquor to remove pigments in the giant salamander skin to obtain the pigment-free giant salamander skin; (4) leaching pigment-free giant salamander skin by using a first acid solution, and then carrying out solid-liquid separation to obtain a crude extract containing collagen; (5) adding a first salt solution into the crude extract to precipitate collagen, and performing solid-liquid separation to obtain primary precipitated collagen; (6) completely dissolving the collagen by using a second acid solution, adding a second hydrochloric solution to separate out the collagen, and performing solid-liquid separation to obtain secondarily separated collagen; (7) dialyzing the secondarily precipitated collagen, and then freeze-drying to obtain a collagen powder product.
In a second aspect, the invention provides a giant salamander collagen product prepared by the method of the first aspect of the invention.
The invention has the following technical effects:
(1) high yield. The method can extract the collagen from the giant salamander skin with the yield of up to 20 percent.
(2) The process is simple. Because the enzymolysis step is not needed, harsh reaction conditions are not needed, and the operation process is simple.
(3) Has comprehensive amino acids and high nutritive value. Many acid processes provide little alanine and acid labile tryptophan that cannot be extracted from giant salamander skin at all, however with the present invention, tryptophan containing up to 0.4 mg/g and alanine 5.0 mg/g can be obtained.
Detailed Description
To make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be described more clearly and completely with reference to the following detailed description of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention belong to the protection of the present invention
The scope of protection.
As described above, the present invention provides in a first aspect a method for extracting collagen from giant salamander skin, wherein the method comprises the steps of: (1) obtaining giant salamander skin; (2) soaking the giant salamander skin by using isopropanol to remove fat in the giant salamander skin to obtain fat-free giant salamander skin; (3) soaking the fat-free giant salamander skin by using alkali liquor to remove pigments in the giant salamander skin to obtain the pigment-free giant salamander skin; (4) leaching pigment-free giant salamander skin by using a first acid solution, and then carrying out solid-liquid separation to obtain a crude extract containing collagen; (5) adding a first salt solution into the crude extract to precipitate collagen, and performing solid-liquid separation to obtain primary precipitated collagen; (6) completely dissolving the collagen by using a second acid solution, adding a second hydrochloric solution to separate out the collagen, and performing solid-liquid separation to obtain secondarily separated collagen; (7) dialyzing the secondarily precipitated collagen, and then freeze-drying to obtain a collagen powder product.
Preferably, in the step (1), the giant salamander skin is 0.4-0.5cm X0.4-0.6 cm small pieces. In some preferred embodiments, all steps are performed in an environment of 2-4 ℃, preferably 4 ℃.
Preferably, in the step (2), the isopropanol is an isopropanol aqueous solution with 8-12 wt%, the material-liquid ratio is 1:10, the soaking is carried out for 24 hours, the liquid is changed every 6 hours, and after the soaking is finished, the giant salamander skin is rinsed to neutral pH by water to obtain the fat-free giant salamander skin.
Preferably, in the step (3), the alkali solution is 0.08-0.12M (i.e. mol/l, the same below) (e.g. 0.09, 0.10 or 0.11M) of sodium hydroxide aqueous solution, the material-to-liquid ratio is 1:20, the soaking time is 24 hours, the alkali solution is changed every 6 hours, and after the soaking is finished, the giant salamander skin is rinsed to neutral pH by water to obtain the pigment-free giant salamander skin.
Preferably, in step (4), the first acid solution is 0.1-0.5M (e.g. 0.2, 0.3 or 0.4M) aqueous citric acid, and the leaching time is 6-24 hours (e.g. 6, 12, 18 or 24 hours); the solid-liquid separation is carried out by the following method: the crude extract was obtained by coarse filtration with gauze and centrifugation of the filtrate at 5000r/min for 20 minutes.
In some preferred embodiments, the aqueous citric acid solution has a concentration of 0.1-0.4M (e.g., 0.1, 0.2, 0.3M, or 0.4M) and the leaching time is 6-18 hours (e.g., 6, 12, or 18 hours); more preferably, the concentration of the aqueous citric acid solution is 0.1-0.3M (e.g., 0.1, 0.2, or 0.3M) and the leaching time is 12 hours, or the concentration of the aqueous citric acid solution is 0.1-0.4M (e.g., 0.1, 0.2, 0.3M, or 0.4M) and the leaching time is 18 hours; most preferably, the concentration of the aqueous citric acid solution is 0.3M and the leaching time is 18 hours.
Preferably, in step (5), the first salt solution is 4.0-6.0M NaCl aqueous solution, preferably 0.5M NaCl aqueous solution, added to the crude extract to make the NaCl concentration of the system 0.9M, left standing for 24 hours for layering, and then centrifuged at 6000r/min for 20 minutes to obtain primary precipitated collagen. In some preferred embodiments, in step (6), the second acid solution is 0.4-0.6M acetic acid aqueous solution, the second salt solution is the same as the first salt solution, and the addition amount is also such that the NaCl concentration of the system is 0.9M, and the secondary precipitation of collagen is obtained.
Preferably, in step (7), the dialysis bag used for dialysis has MW =500, the dialysis solution is distilled water, the dialysis time is 2-4 days, and the frequency of dialysate exchange is 8-12 hours/time. In some embodiments, the methods of the invention comprise the steps of:
(1) obtaining giant salamander skin by the following method: removing residual meat from giant salamander skin, cutting into 0.5cm by 0.5cm, and placing in a refrigerator at 4 deg.C; (2) fat removal: after removing meat residue, putting the cut giant salamander skin into 10 volume percent isopropanol aqueous solution (the material-liquid ratio is 1: 10), soaking for 24 hours, changing the solution once every 6 hours, rinsing for 5 times by tap water after soaking, and rinsing for 5 times by distilled water to obtain fat-free giant salamander skin; (3) removing pigments: soaking the giant salamander skin with the fat removed in a NaOH solution with the concentration of 0.1M at a material-liquid ratio of 1:20 for 24 hours, changing the solution once every 6 hours, rinsing the giant salamander skin with tap water for 5 times after soaking, and rinsing the giant salamander skin with distilled water for 5 times to obtain pigment-free giant salamander skin; (4) extraction: putting the giant salamander skin from which the pigment is removed into a citric acid solution with the concentration of 0.1M, taking out after 6 hours, carrying out coarse filtration by using gauze, centrifuging the filtrate for 20 minutes at 5000r/min, and obtaining a coarse extract after the centrifugation is finished; (5) primary precipitation: adding 5M NaCl into the supernatant to make the concentration of NaCl about 0.9M, standing for 24 hours for layering, and centrifuging at 6000r/min for 20 minutes to obtain primary precipitated collagen; (6) secondary precipitation: adding 0.5M acetic acid into the primary precipitated collagen to dissolve the primary precipitated collagen, taking supernate, adding 5M NaCl to ensure that the concentration of the NaCl is about 0.9M, and obtaining secondary precipitated collagen; (7) dialyzing (MW = 500) for 3d in distilled water, changing the solution every 12 hours; (8) vacuum freeze drying; (9) collecting dry powder; (10) and (6) packaging.
In a second aspect, the invention provides a giant salamander collagen product prepared by the method of the first aspect of the invention.
In some preferred embodiments, the giant salamander collagen product comprises greater than 0.4 mg/g tryptophan and/or greater than 5.0 mg/g alanine.
The giant salamander collagen product prepared by the method is high-purity instant powder, and in some embodiments, the pure low-temperature condition is adopted, so that the nutritional and health-care ingredients of the giant salamander are preserved to the maximum extent, and the giant salamander collagen product is convenient to eat and can be used as an additive of cosmetics and other products.
Examples the technical solutions of the present invention will be illustrated below in the form of examples, but the scope of protection of the present invention is not limited to these examples.
Materials and methods
The reagents used in the examples of the present application are shown in table 1 below.
Table 1 main reagents and their sources.
The main instruments or devices used in the examples of the present application are shown in table 2 below.
TABLE 2 Main instruments or equipment and their sources.
The collection of materials used in this example. Giant salamander skin adopted in embodiment of application
The method is characterized in that the skin of the second-generation giant salamander of the experimental research of Guiyang college biological resource utilization and laboratory feeding control is taken after the second-generation giant salamander is slaughtered, the meat residue on the skin is removed, the second-generation giant salamander is cut into the size of about 0.5cm multiplied by 0.5cm, and the cut meat is placed into a refrigerator for later use. The extraction method comprises the following steps: (1) obtaining giant salamander skin by the following method: removing residual meat from giant salamander skin, cutting into 0.5cm by 0.5cm, and placing in a refrigerator at 4 deg.C;
(2) fat removal: after removing meat residue, putting the cut giant salamander skin into 10 volume percent isopropanol aqueous solution (the material-liquid ratio is 1: 10), soaking for 24 hours, changing the solution once every 6 hours, rinsing for 5 times by tap water after soaking, and rinsing for 5 times by distilled water to obtain fat-free giant salamander skin;
(3) removing pigments: soaking the giant salamander skin with the fat removed in a 0.1M NaOH aqueous solution at a material-liquid ratio of 1:20 for 24 hours, changing the solution once every 6 hours, rinsing the giant salamander skin with tap water for 5 times after soaking, and rinsing the giant salamander skin with distilled water for 5 times to obtain pigment-free giant salamander skin;
(4) extraction: putting the giant salamander skin without the pigment into a citric acid aqueous solution (namely a first acid solution) with the concentration of 0.1M, taking out after 6 hours, carrying out coarse filtration by using gauze, centrifuging the filtrate for 20 minutes at 5000r/min, and obtaining a coarse extract after the centrifugation is finished;
(5) primary precipitation: adding 5M NaCl aqueous solution (first salt solution) into the supernatant to make the concentration of NaCl about 0.9M, standing for 24 hours for layering, and centrifuging at 6000r/min for 20 minutes to obtain primary precipitated collagen;
(6) secondary precipitation: adding 0.5M acetic acid aqueous solution (second acid solution) into the primary precipitated collagen to dissolve the primary precipitated collagen, taking supernate, adding 5M NaCl aqueous solution (second salt solution) to make the concentration of NaCl about 0.9M, and obtaining secondary precipitated collagen;
(7) dialyzing (MW = 500) for 3 days in distilled water, changing the solution every 12 hours;
(8) vacuum freeze drying;
(9) collecting the dry powder and weighing the weight (W) of the obtained dry powder; the experiment was repeated three times and the data are the average of three independent experiments. The yield of collagen was calculated to be 17.93%.
Examples 2 to 20
Collagen was extracted in substantially the same manner as in example 1, except for the contents described in table 3.
Comparative example 1
The same giant salamander skin fragments as used in example 1 were taken and added with pepsin 3%
0.5M acetic acid aqueous solution (material-liquid ratio is 1: 20), leaching for 18 hours at 4 ℃, moving to 5 ℃ after leaching, centrifuging for 20 minutes at 6000rpm, taking supernatant, adding NaCl aqueous solution into the supernatant to enable the NaCl concentration in the solution to be 0.9M, standing for 24 hours, centrifuging for 20 minutes at 8000 rpm at 5 ℃ after layering, taking out and recovering floccule, dialyzing the floccule for 7 days (changing liquid every other day), taking out and freeze-drying to obtain collagen dry powder. The experiment was repeated three times and the data are the average of three independent experiments. The yield of collagen was calculated to be 17.70%.
Comparative example 2
Adding 0.1M NaOH aqueous solution at 4 ℃, soaking for 24 hours (1:20, weight ratio) to remove non-collagen components, washing to neutrality, adding 0.5M acetic acid solution (1:20, weight ratio) to homogenate, magnetically stirring and extracting for 2 days at 4 ℃, centrifuging at 10000rpm for 30 minutes, and obtaining supernatant which is acid-soluble collagen. Adding NaCl into the supernatant to final concentration of 0.9M, centrifuging, collecting precipitate, dissolving in 0.5M acetic acid aqueous solution, dialyzing with 0.1M acetic acid aqueous solution for 1 day, dialyzing with distilled water for 2 days, and lyophilizing to obtain collagen. The experiment was repeated three times and the data are the average of three independent experiments.
Comparative example 3
Substantially the same procedure as in example 1 was followed, except that the steps (2) and (3) were performed in the reverse order, that is, the step (3) in example 1 was performed first and then the step (2) was performed. The experiment was repeated three times and the data are the average of three independent experiments.
Table 3 citric acid concentrations and extraction times used in the different examples and the percentage (%) of collagen obtained relative to that obtained by the enzymatic method.
Relative percentage = collagen obtained in each example/collagen X100% obtained in comparative example 1.
Yield = X100% by weight of collagen/giant salamander skin sample obtained in each example.
Alanine and tryptophan were measured by LC-20AT HPLC. Environmental conditions: the temperature is 18 ℃, the relative humidity is 78 percent, and the content of alanine and tryptophan in the sample is detected according to GB/T16631-2008 high performance liquid chromatography.
As can be seen from the results in Table 3, the yield of collagen comparable to or superior to that obtained by the enzymatic method can be obtained with the present invention, and in some preferred embodiments, a certain amount of tryptophan (which is considered to be unstable to acids and cannot be extracted) can also be extracted with a very small amount of alanine (which is not extracted at all in many extraction methods).
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (6)
1. A method for extracting collagen from giant salamander skin, which is characterized by comprising the following steps:
(1) obtaining giant salamander skin;
(2) soaking the giant salamander skin by using isopropanol to remove fat in the giant salamander skin to obtain fat-free giant salamander skin;
(3) soaking the fat-free giant salamander skin by using alkali liquor to remove pigments in the giant salamander skin to obtain the pigment-free giant salamander skin;
(4) leaching pigment-free giant salamander skin by using a first acid solution, and then carrying out solid-liquid separation to obtain a crude extract containing collagen;
(5) adding a first salt solution into the crude extract to precipitate collagen, and performing solid-liquid separation to obtain primary precipitated collagen;
(6) completely dissolving the collagen by using a second acid solution, adding a second hydrochloric solution to separate out the collagen, and performing solid-liquid separation to obtain secondarily separated collagen;
(7) dialyzing the secondarily precipitated collagen, and then freeze-drying to obtain a collagen powder product;
wherein, in the step (4), the first acid solution is citric acid aqueous solution, the concentration is 0.1 to 0.3M, and the leaching time is 18 hours; the solid-liquid separation is carried out by the following method: coarsely filtering with gauze, and centrifuging the filtrate at 5000r/min for 20 min to obtain coarse extractive solution;
in the step (5), the first salt solution is 4.0-6.0M NaCl aqueous solution, the NaCl concentration of the system is 0.9M after the first salt solution is added into the crude extract, the mixture is kept stand for 24 hours for layering, and then the mixture is centrifuged at 6000r/min for 20 minutes to obtain primary precipitated collagen; and
in the step (6), the second acid solution is 0.4-0.6M acetic acid aqueous solution, the second salt solution is the same as the first salt solution, and the addition amount of the second salt solution enables the NaCl concentration of the system to be 0.9M, so that secondary precipitated collagen is obtained;
the collagen powder product contains tryptophan in an amount of 0.4 mg/g or more and alanine in an amount of 5.0 mg/g or more.
2. The method of claim 1, wherein:
in the step (1), the giant salamander skin is small pieces of 0.4-0.5cm X0.4-0.6 cm; and/or all steps are carried out in an environment of 2-4 ℃.
3. The method of claim 1, wherein: in the step (2), the isopropanol is an isopropanol aqueous solution with the weight percent of 8-12%, the material-liquid ratio is 1:10, the giant salamander skin is soaked for 24 hours, the liquid is changed every 6 hours, and after soaking is finished, the giant salamander skin is rinsed to neutral pH by water, so that the fat-free giant salamander skin is obtained.
4. The method of claim 1, wherein: in the step (3), the step (c),
the alkali liquor is 0.08-0.12M sodium hydroxide aqueous solution, the material-liquid ratio is 1:20, the soaking time is 24 hours, the alkali liquor is changed every 6 hours, and after the soaking is finished, the giant salamander skin is rinsed to neutral pH by water to obtain the pigment-free giant salamander skin.
5. The method according to any one of claims 1 to 4, characterized in that: in step (7), the dialysis bag used for dialysis has MW =500, the dialysis solution is distilled water, the dialysis time is 2-4 days, and the frequency of dialysate exchange is 8-12 hours/time.
6. A giant salamander collagen product produced by the method of any one of claims 1 to 5.
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