CN118146357A - Method for extracting collagen from pigskin, product and application thereof - Google Patents
Method for extracting collagen from pigskin, product and application thereof Download PDFInfo
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- 229920001436 collagen Polymers 0.000 title claims abstract description 77
- 238000000034 method Methods 0.000 title claims abstract description 47
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 111
- CGVLVOOFCGWBCS-RGDJUOJXSA-N n-octyl β-d-thioglucopyranoside Chemical compound CCCCCCCCS[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O CGVLVOOFCGWBCS-RGDJUOJXSA-N 0.000 claims abstract description 24
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 claims abstract description 23
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- 238000000605 extraction Methods 0.000 abstract description 27
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Landscapes
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Abstract
The invention relates to a method for extracting collagen from pigskin, a product and application thereof, wherein the method comprises the following steps: degreasing pigskin, and freeze-drying; hydrolyzing frozen Corii Sus Domestica in pepsin, mixing with octyl thioglucoside and CHAPS, and filtering to obtain filtrate; salting out the filtrate and sodium chloride until flocculent precipitate appears; redissolving the precipitate, sequentially dialyzing in acetic acid and deionized water, and freeze-drying. The invention combines the enzyme method and the acid method extraction method, further improves the purity, prepares the collagen with lower immunogenicity and sensitization risk, and is more suitable for the medical field.
Description
Technical Field
The invention belongs to the technical field of substance extraction, and relates to a method for extracting collagen from pigskin, a product and application thereof.
Background
The pigskin contains water, fat, minerals and protein, wherein the water accounts for 56%, fat about 10%, minerals about 0.1%, and protein about 33%. Wherein the pigskin contains a large amount of protein mainly collagen, and further contains a small amount of albumin, globulin and elastin. The pigskin structure is seen from the longitudinal section and can be divided into three parts of an epidermis layer, a dermis layer and subcutaneous tissues, wherein the dermis layer accounts for 90% of the thickness of the pigskin and is a main component. The dermis layer comprises a fibrous component and a non-fibrous component, the fibrous component consisting essentially of collagen fibers, elastic fibers and reticular fibers, wherein the collagen fibers comprise 99%. With the development of scientific technology, pig skin is increasingly studied, and more attempts and applications are being made on pig skin in many fields. At present, pigskin is developed and applied in the fields of food industry, biomedicine, cosmetics industry and the like besides being applied to the tanning industry.
Four methods for extracting collagen are commonly used, namely an acid method, an alkali method, an enzyme method, a salt method and a hot water extraction method. Different collagen products can be obtained due to different extraction conditions of various methods and different degrees of hydrolysis. The collagen product of pigskin is collagen, gelatin, and collagen polypeptide. Although the three have homology, they have obvious differences in structure, resulting in great differences in their properties. The most obvious difference is that collagen has biological activity and can promote cell growth, while gelatin and polypeptide have no biological activity and can not promote cell growth.
Enzymatic extraction of collagen is currently the ideal method. In the enzymolysis process, non-collagenous peptide at N and C ends of collagen molecules is cut off by protease, but because the triple helix structure is not destroyed, more collagen becomes soluble, and dispersed collagen molecules are gradually dissolved in an acidic environment, thereby achieving the purpose of extracting collagen. The collagen prepared by the enzyme method has higher purity, more stable water solubility and physicochemical property, and the enzymolysis and acid dissolution are synchronously carried out, so the method has the characteristics of quick reaction, short period, no pollution, high purity of the prepared collagen product, good water solubility, stable physicochemical property and the like.
However, collagen in pigskin extracted by the existing method still has the following defects: 1) Animal-derived collagen presents potential viral contamination, immunogenicity, and sensitization risks; 2) The price is high, standardized mass production is difficult to achieve, the cost is reduced, the space is limited, and the price is high; 3) The collagen extracted from animals is insoluble, needs to be dissolved by using an extreme reagent, and has the potential safety hazard of residual strong acid and strong alkali, so that the operation technical requirements on doctors are stricter.
Therefore, in view of the shortcomings and drawbacks of the prior art methods, a new method is used to extract collagen from pigskin to reduce the potential risk of viral contamination, immunogenicity and sensitization.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for extracting collagen from pigskin, and a product and application thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
In a first aspect, the present invention provides a method of extracting collagen from pigskin, the method comprising:
(1) Degreasing pigskin, and freeze-drying;
(2) Hydrolyzing frozen Corii Sus Domestica in pepsin, mixing with octyl thioglucoside and CHAPS, and filtering to obtain filtrate;
(3) Salting out the filtrate and sodium chloride until flocculent precipitate appears;
(4) Redissolving the precipitate, sequentially dialyzing in acetic acid and deionized water, and freeze-drying.
The method for extracting the collagen has higher extraction efficiency, reduces immunogenicity and sensitization risks, and aims to produce a collagen material which can be used in organisms. The use of octyl thioglucoside and CHAPS (4-chloro-2-fluorophenyl-1-methylimidazole) can improve the purity of collagen, and a certain synergistic effect exists between octyl thioglucoside and CHAPS.
Preferably, the degreasing of the pigskin includes immersing the pigskin in a buffer solution for 4-12 hours, for example, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc., and other specific values in the above numerical ranges may be selected, which will not be described in detail herein.
Preferably, the buffer comprises Tris-HCl.
Preferably, the buffer solution further comprises Na 2CO3 and lipase.
Preferably, the freeze drying temperature is-80 to-40 ℃ and the time is 24-72h.
The temperature can be selected from-80 ℃, -75 ℃, -70 ℃, -65 ℃, -60 ℃, -55 ℃, -50 ℃, -45 ℃, -40 ℃ and the like, the time can be selected from 24h, 28h, 32h, 36h, 40h, 44h, 48h, 52h, 56h, 60h, 64h, 68h, 72h and the like, and other specific values in the numerical range can be selected, so that the detailed description is omitted.
Preferably, the temperature of the hydrolysis is 4-37 ℃ and the time is 4-24h.
The temperature may be 4 ℃,5 ℃,10 ℃, 15 ℃, 20 ℃,25 ℃, 30 ℃, 35 ℃, 37 ℃ and the like, the time may be 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours and the like, and other specific point values in the numerical range may be selected, so that the details are not repeated here.
The present invention finds that the temperature and time of hydrolysis are important for the efficiency of collagen extraction.
Preferably, the pH of the hydrolysis is 1.9-2.4, for example, 1.9, 2, 2.1, 2.2, 2.3, 2.4, etc., and other specific values in the above numerical ranges may be selected, which will not be described in detail herein.
Preferably, the pepsin is 1-5% by mass, for example 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% by mass, and other specific values in the above numerical ranges are selected, and will not be described in detail herein.
Preferably, the concentration of the pigskin in the step (2) is 1-20mg/mL, for example 1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL, and other specific values within the above numerical range can be selected, and will not be described in detail herein.
Preferably, the time of mixing with octyl thioglucoside and CHAPS is 22-26h, such as 22h, 22.5h, 23h, 23.5h, 24h, 24.5h, 25h, 25.5h, 26h, etc., and other specific values within the above numerical ranges are selected, and will not be described in detail herein.
Preferably, the concentration of the octyl thioglucoside is 0.5-1.2%, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, etc., and other specific values in the above numerical ranges may be selected, which will not be described in detail herein.
Preferably, the concentration of CHAPS is 0.3-0.8%, for example, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, etc., and other specific values in the above numerical ranges may be selected, and will not be described in detail herein.
Preferably, the final concentration of the sodium chloride is 1-2mol/L, for example, 1mol/L, 1.1mol/L, 1.2mol/L, 1.3mol/L, 1.4mol/L, 1.5mol/L, 1.6mol/L, 1.7mol/L, 1.8mol/L, 1.9mol/L, 2mol/L, etc., and other specific values within the above numerical ranges are selected and will not be described in detail herein.
Preferably, the precipitation is redissolved by adopting 0.5-1mol/L acetic acid solution, for example, 0.5mol/L, 0.6mol/L, 0.7mol/L, 0.8mol/L, 0.9mol/L, 1mol/L and the like, and other specific values in the numerical range can be selected, so that the details are not repeated here.
Preferably, the dialysis time in acetic acid is 12-72 hours, and the concentration of acetic acid is 0.1-0.8mol/L.
The time may be selected from 12h, 16h, 18h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, 48h, 52h, 56h, 60h, 64h, 68h, 72h, etc., and the acetic acid concentration may be selected from 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L, 0.5mol/L, 0.6mol/L, 0.7mol/L, 0.8mol/L, etc., and other specific values within the above numerical ranges may be selected, and will not be described herein.
Preferably, the dialysis time in deionized water is 18-32h, for example, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, etc., and other specific values in the above numerical ranges are selected, which will not be described in detail herein.
In a second aspect, the present invention provides collagen extracted according to the method of the first aspect.
In a third aspect, the present invention provides a use of the collagen according to the second aspect in cosmetic, pharmaceutical, medical device products.
Compared with the prior art, the invention has the following beneficial effects:
The invention combines the enzyme method and the acid method extraction method, further improves the purity, prepares the collagen with lower immunogenicity and sensitization risk, and is more suitable for the medical field. Meanwhile, the combination of octyl thioglucoside and CHAPS is used in the extraction process, so that cell components and microorganism components in residual tissues can be thoroughly destroyed, partial impurity protein components are removed, collagen immunogen generation is reduced from raw materials, and the method can be better applied to medical and cosmetic fields.
Drawings
FIG. 1 is a SDS-PAGE of collagen at various reaction times.
FIG. 2 is a SDS-PAGE of collagen at different reaction temperatures and dialysis buffer.
FIG. 3 is a graph showing the results of DNA content analysis.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Example 1
The embodiment provides a method for extracting collagen, which comprises the following steps:
(1) Treatment of fresh pigskin:
Selecting fresh miniature pig skin at the back of Bama miniature pig, removing hair and dirt, removing subcutaneous fat layer and epidermis layer with knife, leaving dermis layer near epidermis layer, cutting into size of 2cm×2cm, rinsing with deionized water for 3 times, and storing at-20deg.C;
(2) Degreasing
The crushed pigskin is soaked in Tris-NaCl (Tris: 0.05mol/L; naCl:1mol/L; pH 7.5) buffer solution (feed liquid ratio 1:20) for 6h at 4 ℃ and rinsed with deionized water for 3 times. Vacuum freeze drying is carried out for 48 hours;
(3) Extraction of
Hydrolyzing defatted Corii Sus Domestica in 2.0% pepsin solution with pH of 2.2 at 26deg.C for 8 hr, keeping Corii Sus Domestica concentration of 2.5mg/mL, continuously treating with 0.8% octyl thioglucoside and 0.3% CHAPS for 24 hr, and filtering to remove unreacted residue;
(4) Salting out
Adding NaCl to the solution after the reaction until the final concentration is 1.5mol/L, salting out at 4 ℃ until flocculent precipitate exists in the slurry, centrifuging (at 4 ℃ and 10000rpm,15 min), collecting the lower precipitate, redissolving in a solution with the pH value of 2.2 (0.5 mol/L acetic acid), centrifuging to remove insoluble particles (at 4 ℃ and 10000rpm,15 min), and repeating for 3 times.
(5) Dialysis
Dissolving the last salting-out precipitate with solution with pH of 2.2 (0.5 mol/L acetic acid), loading into dialysis bag, dialyzing in 0.1M acetic acid solution for 48 hr, dialyzing in deionized water for 24 hr, and vacuum freeze drying.
Example 2
The embodiment provides a method for extracting collagen, which comprises the following steps:
(1) Treatment of fresh pigskin:
Selecting fresh miniature pig skin at the back of Bama miniature pig, removing hair and dirt, removing subcutaneous fat layer and epidermis layer with knife, leaving dermis layer near epidermis layer, cutting into size of 2cm×2cm, rinsing with deionized water for 3 times, and storing at-20deg.C;
(2) Degreasing
The crushed pigskin is soaked in Tris-NaCl (Tris: 0.05mol/L; naCl:1mol/L; pH 7.5) buffer solution (feed liquid ratio 1:20) for 12h at 4 ℃ and rinsed with deionized water for 3 times. Vacuum freeze drying is carried out for 24 hours;
(3) Extraction of
Hydrolyzing defatted Corii Sus Domestica in 5.0% pepsin acetic acid solution with pH value of 2.4 at 37deg.C for 4 hr, keeping Corii Sus Domestica concentration of 20mg/mL, continuously treating with 0.5% octyl thioglucoside and 0.3% CHAPS for 24 hr, and filtering to remove unreacted residue;
(4) Salting out
Adding NaCl to the solution after the reaction until the final concentration is 1mol/L, salting out at 4 ℃ until flocculent precipitate exists in the slurry, centrifuging (at 4 ℃ and 10000rpm and 15 min), collecting the lower precipitate, redissolving in a solution of acetic acid with pH value of 2.2, centrifuging to remove insoluble particles (at 4 ℃ and 10000rpm and 15 min), and repeating for 3 times.
(5) Dialysis
Dissolving the last salting-out precipitate with 0.8mol/L acetic acid solution, loading into dialysis bag, dialyzing in 0.3M acetic acid solution for 72 hr, dialyzing in deionized water for 24 hr, and vacuum freeze drying.
Example 3
The embodiment provides a method for extracting collagen, which comprises the following steps:
(1) Treatment of fresh pigskin:
Selecting fresh miniature pig skin at the back of Bama miniature pig, removing hair and dirt, removing subcutaneous fat layer and epidermis layer with knife, leaving dermis layer near epidermis layer, cutting into size of 2cm×2cm, rinsing with deionized water for 3 times, and storing at-20deg.C;
(2) Degreasing
The crushed pigskin is soaked in Tris-NaCl (Tris: 0.05mol/L; naCl:1mol/L; pH 7.5) buffer solution (feed liquid ratio 1:20) for 4h at 4 ℃ and rinsed with deionized water for 3 times. Vacuum freeze drying is carried out for 72 hours;
(3) Extraction of
Hydrolyzing defatted Corii Sus Domestica in 1.0% pepsin acetic acid solution at 4deg.C and pH of 1.9 for 4 hr to obtain Corii Sus Domestica concentration of 1mg/mL, continuously treating with 0.5% octyl thioglucoside and 0.8% CHAPS for 24 hr, and filtering to remove unreacted residue;
(4) Salting out
Adding NaCl to the solution after the reaction until the final concentration is 1mol/L, salting out at 4 ℃ until flocculent precipitate exists in the slurry, centrifuging (at 4 ℃ and 10000rpm and 15 min), collecting the lower precipitate, redissolving in a solution of acetic acid with pH value of 2.2, centrifuging to remove insoluble particles (at 4 ℃ and 10000rpm and 15 min), and repeating for 3 times.
(5) Dialysis
Dissolving the last salting-out precipitate with 1mol/L acetic acid solution, loading into dialysis bag, dialyzing in 0.8M acetic acid solution for 12 hr, dialyzing in deionized water for 24 hr, and vacuum freeze drying.
Example 4
The present example provides a collagen extraction method, which is different from example 1 only in that the step (3) is "taking defatted pigskin, hydrolyzing in 2.0% pepsin (1:3000) solution with pH value of 2.2 (0.5 mol/L acetic acid) at 26 ℃ for 4 hours, the pigskin concentration is 2.5mg/mL, then continuously treating for 24 hours with 0.8% octyl thioglucoside and 0.3% CHAPS, filtering to remove unreacted residues", and other operations remain unchanged.
Example 5
The present example provides a collagen extraction method, which is different from example 1 only in that the step (3) is "taking defatted pigskin, hydrolyzing in 2.0% pepsin (1:3000) solution with pH value of 2.2 (0.5 mol/L acetic acid) at 26 ℃ for 12 hours, then continuously treating with 0.8% octyl thioglucoside and 0.3% CHAPS for 24 hours, filtering to remove unreacted residues", and other operations remain unchanged.
Example 6
The present example provides a collagen extraction method, which is different from example 1 only in that the step (3) is "taking defatted pigskin, hydrolyzing in 2.0% pepsin (1:3000) solution with pH value of 2.2 (0.5 mol/L acetic acid) at 26 ℃ for 16h, the pigskin concentration is 2.5mg/mL, then continuously treating for 24h with 0.8% octyl thioglucoside and 0.3% CHAPS, filtering to remove unreacted residues", and other operations remain unchanged.
Example 7
The present example provides a collagen extraction method, which is different from example 1 only in that the step (3) is "taking defatted pigskin, hydrolyzing in 2.0% pepsin (1:3000) solution with pH value of 2.2 (0.5 mol/L acetic acid) at 26 ℃ for 20 hours, then continuously treating with 0.8% octyl thioglucoside and 0.3% CHAPS for 24 hours, filtering to remove unreacted residues", and other operations remain unchanged.
Example 8
The present example provides a collagen extraction method, which is different from example 1 only in that the step (3) is "taking defatted pigskin, hydrolyzing in 2.0% pepsin (1:3000) solution with pH value of 2.2 (0.5 mol/L acetic acid) at 26 ℃ for 24 hours, then continuously treating with 0.8% octyl thioglucoside and 0.3% CHAPS for 24 hours, filtering to remove unreacted residues", and other operations remain unchanged.
Example 9
The present example provides a collagen extraction method, which is different from example 1 only in that the step (3) is "taking defatted pigskin, hydrolyzing in 2.0% pepsin (1:3000) solution with pH value of 2.2 (0.5 mol/L acetic acid) at 26 ℃ for 8 hours, the pigskin concentration is 1mg/mL, then continuously treating for 24 hours with 0.8% octyl thioglucoside and 0.3% CHAPS, and filtering to remove unreacted residues", and other operations remain unchanged.
Example 10
The present example provides a collagen extraction method, which is different from example 1 only in that the step (3) is "taking defatted pigskin, hydrolyzing in 2.0% pepsin (1:3000) solution with pH value of 2.2 (0.5 mol/L acetic acid) at 26 ℃ for 8 hours, the pigskin concentration is 5mg/mL, then continuously treating for 24 hours with 0.8% octyl thioglucoside and 0.3% CHAPS, and filtering to remove unreacted residues", and other operations remain unchanged.
Example 11
The present example provides a collagen extraction method, which is different from example 1 only in that the step (3) is "taking defatted pigskin, hydrolyzing in 2.0% pepsin (1:3000) solution with pH value of 2.2 (0.5 mol/L acetic acid) at 26 ℃ for 8 hours, the pigskin concentration is 10mg/mL, then continuously treating for 24 hours with 0.8% octyl thioglucoside and 0.3% CHAPS, and filtering to remove unreacted residues", and other operations remain unchanged.
Example 12
The present example provides a collagen extraction method, which is different from example 1 only in that the step (3) is "taking defatted pigskin, hydrolyzing in 2.0% pepsin (1:3000) solution with pH value of 2.2 (0.5 mol/L acetic acid) at 4 ℃ for 8 hours, the pigskin concentration is 10mg/mL, then continuously treating for 24 hours with 0.8% octyl thioglucoside and 0.3% CHAPS, and filtering to remove unreacted residues", and other operations remain unchanged.
Example 13
This example provides a collagen extraction method which differs from example 1 only in that step (5) is "the last salting-out precipitation is dissolved with a solution of pH 2.2 (0.5 mol/L acetic acid), put into a dialysis bag, dialyzed in 0.02M sodium phosphate solution for 48h, dialyzed in deionized water for 24h, followed by vacuum freeze-drying", and the other operations remain unchanged.
Comparative example 1
The comparative example provides a collagen extraction method which is different from example 1 only in that the step (3) is that the defatted pigskin is taken and hydrolyzed in 2.0% pepsin (1:3000) solution with pH value of 2.2 (0.5 mol/L acetic acid) at 26 ℃ for 8 hours, the pigskin concentration is 10mg/mL, and then the pigskin is continuously treated for 24 hours by using 1.1% octyl thioglucoside, and unreacted residues are removed by filtration, and other operations are kept unchanged.
Comparative example 2
The comparative example provides a collagen extraction method differing from example 1 only in that step (3) is "taking defatted pigskin, hydrolyzing in 2.0% pepsin (1:3000) solution with pH of 2.2 (0.5 mol/L acetic acid) at 26 ℃ for 8 hours, the pigskin concentration being 10mg/mL, then continuously treating for 24 hours with 1.1% CHAPS, and then filtering to remove unreacted residues", and the other operations remain unchanged.
Comparative example 3
The comparative example provides a collagen extraction method differing from example 1 only in that step (3) was carried out without continuous treatment with 0.8% octyl thioglucoside and 0.3% chaps, and unreacted residues were removed by directly pepsin and then filtered, and the other operations were kept unchanged.
Comparative example 4
This comparative example provides a collagen extraction method which differs from example 1 only in that step (2) is not included and other operations remain unchanged.
Test example 1
Structure and purity identification
The purity detection calculation is carried out by adopting a polyacrylamide gel electrophoresis and coomassie blue staining method and a gel imaging system, the structure identification and purity analysis are carried out on the gel, and the purity result is shown in Table 2 in detail.
As shown in fig. 1 and 2, the sizes of α1 and α2 chains of collagen are about 110kda and 130kda, and the size of β chains of collagen is about 200kda, and the reason for having two chains is that the speed of β chains having a terminal peptide structure and an endless peptide structure during gel electrophoresis is different, so that there are two bands. FIG. 1 is a SDS-PAGE chart of collagen at different reaction times, and as can be seen from FIG. 2, the change of reaction temperature and dialysis buffer does not affect the extracted collagen component, and by changing the dialysis conditions, we compare whether the collagen component is changed or not under the condition that 0.1M acetic acid and 0.02M phosphate are used as buffer solutions, and as a result, the results show that when the two buffers are used as dialysis solutions, no obvious effect is exerted on collagen, and the reaction system selects acetic acid as a reaction extracting solution, and then uses acetic acid as a dialysis buffer, wherein the buffer is the same as the extracting solution, and the same solution can reduce the solution types in the reaction, and better sensitiveness is reduced.
Test example 2
Analysis of DNA content
Test sample: collagen obtained in example 1 and comparative example 4
The testing method comprises the following steps: after the residual DNA was extracted using the DNA extraction kit, the concentration was measured with an ultra-micro uv spectrophotometer. After the residual DNA was extracted using the DNA extraction kit, the concentration was measured with an ultra-micro uv spectrophotometer.
The electrophoresis results are shown in FIG. 3, the DNA content before and after degreasing in the collagen extraction process has obvious change, the content before degreasing is 94ng/mg, and the content after degreasing is 3.4ng/mg.
The decrease in DNA content can reduce sensitization to some extent, and from the above results, it is clear that the DNA content is significantly reduced after degreasing, and thus the sensitization of collagen is significantly reduced by degreasing.
Test example 3
Measurement of collagen extraction Rate
The testing method comprises the following steps: the principle of specific combination of sirius red and collagen is utilized, the red compound obtained after eluting and centrifuging by 0.1M NaOH is used for determining the collagen content by measuring absorbance under the condition of the wavelength of 540 nm.
3.1 Preparation of standard curve
Taking 0, 10, 20, 30, 40, 50, 60 and 100ul of collagen standard solution (1 mg/mL), adding 1mL of 100 mu mol/L sirius red staining solution, standing at room temperature for 30min, centrifuging 12000r/min, and 30min. The supernatant was discarded, and 1mL of 0.5mol/L acetic acid was added to centrifuge, 12000r/min,30min. The supernatant was discarded and the pellet was eluted with 2mL of 0.1 mol/LNaOH. Absorbance was measured at 540nm and a standard curve was drawn.
3.2 Measurement of collagen content of sample to be measured
The collagen extract was examined by referring to a standard curve preparation method, and quantified by a standard curve.
TABLE 1
Test example 4
Immunogenicity evaluation
According to the method for evaluating immunogenicity of medical devices YY/T1465.2-2016, part 2 of the method for evaluating immunogenicity of medical devices: in the method of serum immunoglobulin and complement component assay, a positive control group (BSA), a negative control group and a test sample group are provided, wherein the test sample group is collagen prepared by the embodiment of the invention, and each group comprises 3 mice. The positive control group was prepared by mixing 3mg of BSA with 9mLPBS (pH 7.4) and then with CFA in a volume of 1:1 to give an emulsion, and subcutaneously injecting 0.12mL into the back of each mouse once a week. Test sample group, taking the collagen prepared in quantitative example, and implanting into subcutaneous tissue of back of a mouse. The negative control group was subjected to a sham operation treatment without using a specimen sample. After 2 weeks, the serum of the mice was taken and the content of serum immunoglobulin and serum complement components were determined using a mouse immunoglobulin G (IgG) ELISA kit and a mouse complement component C3a (C3 a) ELISA detection kit, respectively. The higher the IgG and C3a content, the stronger the immune response. The test results were averaged and the results are shown in table 2.
TABLE 2
According to the table data, when the preparation method provided by the invention is adopted for cell removal treatment and the enzyme and nonionic detergent composition are used in combination, the collagen has the highest purity and lower immunogenicity; when decellularized with an enzyme and a nonionic detergent, collagen purity decreases and immunogenicity increases.
The applicant states that the present invention is described by way of the above examples as a method of extracting collagen from pigskin and its products and applications, but the present invention is not limited to, i.e. it is not meant that the present invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. A method for extracting collagen from pigskin, the method comprising:
(1) Degreasing pigskin, and freeze-drying;
(2) Hydrolyzing frozen Corii Sus Domestica in pepsin, mixing with octyl thioglucoside and CHAPS, and filtering to obtain filtrate;
(3) Salting out the filtrate and sodium chloride until flocculent precipitate appears;
(4) Redissolving the precipitate, sequentially dialyzing in acetic acid and deionized water, and freeze-drying.
2. The method of claim 1, wherein degreasing the pigskin comprises immersing the pigskin in a buffer for 4-12 hours;
Preferably, the buffer comprises Tris-HCl.
3. The method according to claim 1 or 2, wherein the freeze-drying is carried out at a temperature of-80 to-40 ℃ for a time of 24 to 72 hours.
4. A process according to any one of claims 1 to 3, wherein the hydrolysis is carried out at a temperature of 4 to 37 ℃ for a period of 4 to 24 hours;
Preferably, the pH of the hydrolysis is 1.9-2.4;
Preferably, the mass percentage of the pepsin is 1-5%;
Preferably, the concentration of pigskin in step (2) is 1-20mg/mL;
Preferably, the time of mixing with octyl thioglucoside and CHAPS is 22-26 hours;
preferably, the concentration of the octyl thioglucoside is 0.5-1.2%;
preferably, the concentration of CHAPS is 0.3-0.8%.
5. The method according to any one of claims 1 to 4, wherein the final concentration of sodium chloride is 1-2mol/L.
6. The method according to any one of claims 1 to 5, wherein the precipitate is reconstituted with 0.5 to 1mol/L acetic acid solution.
7. The method according to any one of claims 1 to 6, wherein the dialysis is performed in acetic acid for a period of 12 to 72 hours and the concentration of acetic acid is 0.1 to 0.8mol/L.
8. The method of any one of claims 1-7, wherein the dialysis in deionized water is for 18-32 hours.
9. Collagen extracted according to the method of any one of claims 1-8.
10. Use of the collagen according to claim 9 in cosmetic, pharmaceutical, medical device products.
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