CN106946988A - A kind of extracting method of ox heel string collagen - Google Patents
A kind of extracting method of ox heel string collagen Download PDFInfo
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- CN106946988A CN106946988A CN201710223675.6A CN201710223675A CN106946988A CN 106946988 A CN106946988 A CN 106946988A CN 201710223675 A CN201710223675 A CN 201710223675A CN 106946988 A CN106946988 A CN 106946988A
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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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Abstract
The invention discloses a kind of extracting method of ox heel string collagen, the step of it includes pre-treatment, extracts, precipitates, saltouing, purifying, drying and preserving collagen.The extracting method of the ox heel string collagen of the present invention prepares the method being combined using enzymolysis and high performance liquid chromatography can isolate and purify I-type collagen from yak heel string, and this method has high efficiency, high selectivity and easy amplification.
Description
Technical field
Method of the present invention is related to a kind of extracting method of ox heel string collagen.
Background technology
Current collagen is main to be extracted from pigskin, ox-hide, fish-skin and animal skeleton.The collagen egg in animal skin source
White contaminated probability is high, and animal skeleton source collagen recovery rate is low.Existing extraction system：Organic acid soln system, stomach egg
White enzyme-acid solution system and neutral salt solution system, wherein pepsin-acid solution system acquisition rate highest.Common purification
Mode is simple using step column chromatography progress, and it is not high that the method has production cycle length, low separation efficiency, product purity
The shortcomings of.Long separation cycle and low separative efficiency not only increase the production cost of collagen, and make many resources
It can not be effectively utilized.
The content of the invention
The purpose of method of the present invention is to provide a kind of extracting method of ox heel string collagen, can be separated from yak heel string
I-type collagen is purified, this method has high efficiency, high selectivity and easy amplification.
In order to solve the above technical problems, method of the present invention is adopted the following technical scheme that：
A kind of extracting method of ox heel string collagen, comprises the following steps：
A, pre-treatment：Fresh ox heel string removes soft tissue, rejected after manadesma, fat, and pulverizer is crushed to 0.2~0.5 mm sizes
Tissue block, 5 min, mass fraction 5%NaHCO are soaked by mass fraction 20%NaCL solution3Solution soaks 30 min, removes blood
Clear to wait composition, sterilization, later operation is all completed under sterile working；Chloroform will be put into the tissue fritter after distillation water washing
It is 2 with ethanol volume ratio:1 solution carries out degreasing to it, and degreasing 2-6 h, distilled water is washed 3 times；With NaCl containing 4.5mol/L
0.05mol/L Tris-HCl buffer solutions（pH 7.5）Fully washing 12-36h, to remove the compositions such as lipid and serum, low temperature
Centrifugation；
B, extracting：The 0.5mol/L glacial acetic acid for adding 1L 500mg containing pepsin will be precipitated, stir and maintain low temperature, extracted
72h, 10 min are centrifuged per 10000g, remove precipitation；
C, precipitation：0.5mol/L glacial acetic acid is added in supernatant, it is 2.5~3.0 to make pH value of solution, added in usual every liter of extract solution
Enter 30ml glacial acetic acid, be sufficiently stirred for magnetic stirring apparatus, precipitation is abandoned after centrifugation；
D, saltout：NaCl is added in supernatant to 4.4mol/L, is stirred overnight, precipitation is dissolved in 0.5mol/L by low-temperature centrifugation
In glacial acetic acid, then acid of being saltoutd repeatedly with 2.4mol/L, 1.7mol/L and 1.0mol/L NaCl solution step by step is molten, finally obtains
Supernatant is collagen crude extract；
E, by collagen crude extract freeze-drying obtain collagen crude extract dried frozen aquatic products, under low temperature preserve
F, high-efficient liquid phase chromatogram purification：By the ultrapure water dissolves of collagen dried frozen aquatic products, through membrane filtration, filtrate loading is taken to carry out chromatogram
Separation.
Further, low-temperature centrifugation condition is 8000r/min, 30min, 4 DEG C in the step A.
Further, chromatographic condition is in the step F：The SB-C18 of ZORBAX 300 partly prepare chromatographic column, and 9.4mm ×
25cm, mobile phase：A is water（85%）, B is methanol（15%）；Eluted using linear gradient；Flow velocity：1ml.min-1 ；Sample size：
500uL；Detection wavelength：220nm；Column temperature：Room temperature, collects main peak, for physicochemical property identification.
Further, filter membrane is 0.45um filter membranes in the step F.
Further, degreasing time is 4h in the step A.
Further, with the 0.05mol/L Tris-HCl buffer solutions of the NaCl containing 4.5mol/L in the step A（pH
7.5）Abundant wash time is 24h.
Further, the cryogenic temperature for stirring and maintaining in the step B is 4 DEG C.
Further, the pepsin 1mg is containing 2500 active units.
Further, the preparation method of the buffer solution is：The Tris of 6.06 parts by weight is dissolved in 40 parts by weight
ddH2In O, pH is adjusted to 7.5 with 4mol/L HCL, then add 10 parts by weight ddH2O, 4 DEG C of preservations.
Present invention also offers a kind of ox heel string collagen protein sponge, its preparation method comprises the following steps：
（1）Collagen Dai Bai after high-efficient liquid phase chromatogram purification in step F is stirred, bubble is centrifuged off, regulation pH value to
6, dilution is defined by the swollen liquid of collagen that 50-70ml is made in 1g consumption ox heel strings；
（2）The swollen liquid of collagen is poured into mould, pre-freeze 24h at -50 DEG C, glue is obtained through -30 DEG C to -50 DEG C freeze-drying 24h
Former protein sponge；
（3）By collagen protein sponge 60Co radiosterilizations, encapsulation, -4 DEG C save backup.
It is preferred that, step（1）The 60ml swollen liquid of collagen is made in middle 1g consumptions ox heel string；Step（2）In, it is cold through -40 DEG C
Freeze dry 24h and obtain collagen protein sponge.
The collagen protein sponge is a kind of absorbable surgical hemostasis, the auxiliary material of leak stopping, reliability safe and convenient to use.
Compared with prior art, Advantageous Effects of the invention：
The collagen in ox heel string source is not easy pollution compared with the collagen of skin-derived, more cleans, and recovery rate is higher；Tropocollagen molecule
Main part is shaft-like triple-helix structure, the effect of energy quite tolerant pepsin, but the globulin structure energy quilt at molecule two ends
Pepsin selective hydrolysis.When pH value is 2.5~3.0, pepsin can be successfully by terminal peptide from complete collagen master
Cut down on body molecule, the huge molecular structure of the collagen being so crosslinked is destroyed, approximate complete tropocollagen molecule just can
Separate and be extracted from collagenous fibres, so enzymatic isolation method extracts the yield highest of collagen；With high performance liquid chromatography point
Advantage from purifying I-type collagen is that sensitivity is high it will be apparent that it has the post effect higher than common column chromatography, point
From mild condition, the typically no specimen breakdown during separation detection, it is easy to which sample is reclaimed, and operation automation；Utilize
Enzymolysis and high performance liquid chromatography prepare the method being combined can isolate and purify I-type collagen, this method from yak heel string
With high efficiency, high selectivity and easy amplification.
Embodiment
A kind of extracting method of ox heel string collagen, comprises the following steps：
1st, pre-treatment：Fresh ox heel string removes soft tissue, reject 100 g are weighed after manadesma, fat, and pulverizer is crushed to 0.2~
0.5 mm size tissue blocks, 5 min, mass fraction 5%NaHCO are soaked by mass fraction 20%NaCL330 min are soaked, blood is removed
Clear to wait composition, sterilization, later operation is all completed under sterile working.Chlorine will be put into the tissue fritter after distillation water washing
Imitative/ethanol（2:1）Solution carries out degreasing to it, and the h of degreasing 4, distilled water is washed 3 times.With the NaCl's containing 4.5mol/L
0.05mol/L Tris-HCl buffer solutions（pH 7.5）Fully washing 24h, to remove the compositions such as lipid and serum, low-temperature centrifugation
（8000r/min, 30min, 4 DEG C）；Buffer liquid and preparation method thereof：6.06g Tris are dissolved in 40 ml ddH2In O, 4mol/ is used
L HCL adjust pH to 7.5, then add ddH2O total amount is to 50ml（Add 10mlddH2O）, 4 DEG C of preservations.
2nd, extract：The 0.5mol/L glacial acetic acid for adding 1L 500mg containing pepsin will be precipitated, stirs and maintains 4 DEG C, carry
Take 72h, every 10000 g to centrifuge 10 min, remove precipitation；
3rd, precipitate：0.5mol/L glacial acetic acid is added in supernatant, it is 2.5~3.0 to make pH value of solution, added in usual every liter of extract solution
Enter 30ml glacial acetic acid, be sufficiently stirred for magnetic stirring apparatus, precipitation is abandoned after centrifugation；
4th, saltout：NaCl is added in supernatant to 4.4mol/L, is stirred overnight, precipitation is dissolved in 0.5mol/L by low-temperature centrifugation
In glacial acetic acid, then saltoutd repeatedly with 2.4mol/L, 1.7mol/L and 1.0mol/L NaCl step by step, acid it is molten.What is finally obtained is upper
Clear liquid is collagen crude extract；
5th, the freeze-drying of collagen crude extract is obtained into collagen crude extract dried frozen aquatic products, in preservation under low temperature；
6th, high-efficient liquid phase chromatogram purification：By the ultrapure water dissolves of collagen dried frozen aquatic products, through 0.45um membrane filtrations, filtrate loading is taken to enter
Row chromatographic isolation；Chromatographic condition：The SB-C18 of ZORBAX 300 partly prepare chromatographic column（9.4mm×25cm）, mobile phase：A is water
（85%）, B is methanol（15%）；Eluted using linear gradient；Flow velocity：1ml.min-1 ；Sample size：500uL；Detection wavelength：
220nm；Column temperature：Room temperature, collects main peak, for physicochemical property identification.
Present invention also offers a kind of ox heel string collagen protein sponge, its preparation method comprises the following steps：
（1）Collagen Dai Bai after high-efficient liquid phase chromatogram purification in step F is stirred, bubble is centrifuged off, uses 0.05mol/
NaOH solution adjusts pH value to 6, is diluted with ultra-pure water, is defined by the swollen liquid of collagen that 60ml is made in 1g consumption ox heel strings；
（2）The swollen liquid of collagen is poured into mould, pre-freeze 24h at -50 DEG C, collagen sea is obtained through -40 DEG C of freeze-drying 24h
It is continuous；
（3）By collagen protein sponge 60Co radiosterilizations, encapsulation, -4 DEG C save backup.
The collagen protein sponge is a kind of absorbable surgical hemostasis, the auxiliary material of leak stopping, reliability safe and convenient to use.
Embodiment described above is only that the preferred embodiment of method of the present invention is described, not to the scope of method of the present invention
It is defined, on the premise of method design spirit of the present invention is not departed from, technology of the those of ordinary skill in the art to method of the present invention
In various modifications and improvement that scheme is made, the protection domain that claims of the present invention determination all should be fallen into.
Claims (10)
1. a kind of extracting method of ox heel string collagen, it is characterised in that comprise the following steps：
A, pre-treatment：Fresh ox heel string removes soft tissue, rejected after manadesma, fat, and pulverizer is crushed to 0.2~0.5 mm sizes
Tissue block, 5 min, mass fraction 5%NaHCO are soaked by mass fraction 20%NaCL solution3Solution soaks 30 min, removes blood
Clear to wait composition, sterilization, later operation is all completed under sterile working；Chloroform will be put into the tissue fritter after distillation water washing
With ethanol volume ratio 2:1 solution carries out degreasing to it, and degreasing 2-6 h, distilled water is washed 3 times；With the NaCl's containing 4.5mol/L
PH is 7.5 0.05mol/L Tris-HCl buffer solutions, 12-36h is fully washed, to remove the compositions such as lipid and serum, low temperature
Centrifugation；
B, extracting：The 0.5mol/L glacial acetic acid for adding 1L 500mg containing pepsin will be precipitated, stir and maintain low temperature, extracted
72h, 10 min are centrifuged per 10000g, remove precipitation；
C, precipitation：0.5mol/L glacial acetic acid is added in supernatant, it is 2.5~3.0 to make pH value of solution, added in usual every liter of extract solution
Enter 30ml glacial acetic acid, be sufficiently stirred for magnetic stirring apparatus, precipitation is abandoned after centrifugation；
D, saltout：NaCl is added in supernatant to 4.4mol/L, is stirred overnight, precipitation is dissolved in 0.5mol/L by low-temperature centrifugation
In glacial acetic acid, then acid of being saltoutd repeatedly with 2.4mol/L, 1.7mol/L and 1.0mol/L NaCl solution step by step is molten, finally obtains
Supernatant is collagen crude extract；
E, by collagen crude extract freeze-drying obtain collagen crude extract dried frozen aquatic products, under low temperature preserve
F, high-efficient liquid phase chromatogram purification：By the ultrapure water dissolves of collagen dried frozen aquatic products, through membrane filtration, filtrate loading is taken to carry out chromatogram
Separation.
2. the extracting method of ox heel string collagen according to claim 1, it is characterised in that：Low temperature in the step A
Centrifugal condition is 8000r/min, 30min, 4 DEG C.
3. the extracting method of ox heel string collagen according to claim 1, it is characterised in that：Chromatogram in the step F
Condition is：The SB-C18 of ZORBAX 300 partly prepare chromatographic column, 9.4mm × 25cm, mobile phase：A is water（85%）, B is methanol
（15%）；Eluted using linear gradient；Flow velocity：1ml.min-1 ；Sample size：500uL；Detection wavelength：220nm；Column temperature：Room temperature,
Main peak is collected, for physicochemical property identification.
4. the extracting method of ox heel string collagen according to claim 1, it is characterised in that：Stirred in the step B
And the cryogenic temperature maintained is 4 DEG C, filter membrane is 0.45um filter membranes in the step F.
5. the extracting method of ox heel string collagen according to claim 1, it is characterised in that：Degreasing in the step A
Time is 4h.
6. the extracting method of ox heel string collagen according to claim 1, it is characterised in that：With containing in the step A
4.5mol/L NaCl 0.05mol/L Tris-HCl buffer solutions（pH 7.5）Abundant wash time is 24h.
7. the extracting method of ox heel string collagen according to claim 1, it is characterised in that：The pepsin 1mg
Containing 2500 active units.
8. the extracting method of ox heel string collagen according to claim 1, it is characterised in that：The preparation of the buffer solution
Method is：The Tris of 6.06 parts by weight is dissolved in the ddH of 40 parts by weight2In O, pH is adjusted to 7.5 with 4mol/L HCL, then
Plus 10 parts by weight ddH2O, 4 DEG C of preservations.
9. a kind of preparation method for consuming ox heel string collagen protein sponge, it is characterised in that comprise the following steps：
（1）Collagen Dai Bai after high-efficient liquid phase chromatogram purification in step F is stirred, bubble is centrifuged off, regulation pH value to
6, dilution is defined by the swollen liquid of collagen that 50-70ml is made in 1g consumption ox heel strings；
（2）The swollen liquid of collagen is poured into mould, pre-freeze 24h at -50 DEG C, glue is obtained through -30 DEG C to -50 DEG C freeze-drying 24h
Former protein sponge；
（3）By collagen protein sponge 60Co radiosterilizations, encapsulation, -4 DEG C save backup.
10. the preparation method of consumption ox heel string collagen protein sponge according to claim 1, it is characterised in that step（1）
In, the 60ml swollen liquid of collagen is made in 1g consumption ox heel strings；Step（2）In, obtain collagen sea through -40 DEG C of freeze-drying 24h
It is continuous.
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Cited By (13)
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| CN107236778A (en) * | 2017-08-08 | 2017-10-10 | 北京华信佳音医疗科技发展有限责任公司 | A kind of extracting method of water-soluble collagen |
| CN107354192A (en) * | 2017-08-18 | 2017-11-17 | 广东医科大学 | A kind of method for purifying NTx albumen |
| CN107937462A (en) * | 2017-11-16 | 2018-04-20 | 金华市艾力生物科技有限公司 | A kind of preparation method of yak collagen |
| CN108853571A (en) * | 2018-07-23 | 2018-11-23 | 天津市长江医疗器械有限公司 | A kind of collagen protein sponge and its preparation process |
| CN109566845A (en) * | 2018-12-03 | 2019-04-05 | 甘肃农业大学 | A kind of food-borne beef tendon ace inhibitory peptide, fruits and vegetables chewable tablets and preparation method thereof |
| CN109731136A (en) * | 2019-01-11 | 2019-05-10 | 四川大学 | A kind of preparation method and application of cartilage-inducing matrix material |
| CN109810187A (en) * | 2017-11-20 | 2019-05-28 | 中国科学院大连化学物理研究所 | A kind of extraction method and application of animal hoof tendon collagen |
| CN112410392A (en) * | 2020-11-11 | 2021-02-26 | 武汉盛世伟度生物科技有限公司 | Extraction method and application of type I collagen |
| CN113520900A (en) * | 2021-04-13 | 2021-10-22 | 甘肃天际生物科技有限公司 | Hypoallergenic and anti-aging yak collagen composition and application thereof |
| CN114272361A (en) * | 2021-12-17 | 2022-04-05 | 锐腾(苏州)生物科技有限公司 | Composition and application method of collagen molecular monomer skin coating liquid |
| CN116983459A (en) * | 2023-07-14 | 2023-11-03 | 湖南贝耐特生物科技有限公司 | Preparation method of low-immunogenicity absorbable suture based on tendon of castoreum |
| CN116982667A (en) * | 2023-08-10 | 2023-11-03 | 西北农林科技大学 | Method for improving gel strength and water retention of sheep by-product collagen |
| CN117205306A (en) * | 2023-07-31 | 2023-12-12 | 成都维德医疗器械有限责任公司 | Collagen extract, composition, preparation method and application |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107236778A (en) * | 2017-08-08 | 2017-10-10 | 北京华信佳音医疗科技发展有限责任公司 | A kind of extracting method of water-soluble collagen |
| CN107354192A (en) * | 2017-08-18 | 2017-11-17 | 广东医科大学 | A kind of method for purifying NTx albumen |
| CN107937462A (en) * | 2017-11-16 | 2018-04-20 | 金华市艾力生物科技有限公司 | A kind of preparation method of yak collagen |
| CN109810187A (en) * | 2017-11-20 | 2019-05-28 | 中国科学院大连化学物理研究所 | A kind of extraction method and application of animal hoof tendon collagen |
| CN108853571A (en) * | 2018-07-23 | 2018-11-23 | 天津市长江医疗器械有限公司 | A kind of collagen protein sponge and its preparation process |
| CN109566845A (en) * | 2018-12-03 | 2019-04-05 | 甘肃农业大学 | A kind of food-borne beef tendon ace inhibitory peptide, fruits and vegetables chewable tablets and preparation method thereof |
| CN109731136A (en) * | 2019-01-11 | 2019-05-10 | 四川大学 | A kind of preparation method and application of cartilage-inducing matrix material |
| CN109731136B (en) * | 2019-01-11 | 2020-12-25 | 四川大学 | Preparation method and application of cartilage inductive matrix material |
| CN112410392A (en) * | 2020-11-11 | 2021-02-26 | 武汉盛世伟度生物科技有限公司 | Extraction method and application of type I collagen |
| CN113520900A (en) * | 2021-04-13 | 2021-10-22 | 甘肃天际生物科技有限公司 | Hypoallergenic and anti-aging yak collagen composition and application thereof |
| CN114272361A (en) * | 2021-12-17 | 2022-04-05 | 锐腾(苏州)生物科技有限公司 | Composition and application method of collagen molecular monomer skin coating liquid |
| CN116983459A (en) * | 2023-07-14 | 2023-11-03 | 湖南贝耐特生物科技有限公司 | Preparation method of low-immunogenicity absorbable suture based on tendon of castoreum |
| CN117205306A (en) * | 2023-07-31 | 2023-12-12 | 成都维德医疗器械有限责任公司 | Collagen extract, composition, preparation method and application |
| CN117205306B (en) * | 2023-07-31 | 2024-05-14 | 成都维德医疗器械有限责任公司 | Collagen extract, composition, preparation method and application |
| CN116982667A (en) * | 2023-08-10 | 2023-11-03 | 西北农林科技大学 | Method for improving gel strength and water retention of sheep by-product collagen |
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