CN109810187A - A kind of extracting method and application of animal tendon tendon collagen - Google Patents
A kind of extracting method and application of animal tendon tendon collagen Download PDFInfo
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- CN109810187A CN109810187A CN201711154860.0A CN201711154860A CN109810187A CN 109810187 A CN109810187 A CN 109810187A CN 201711154860 A CN201711154860 A CN 201711154860A CN 109810187 A CN109810187 A CN 109810187A
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Abstract
The present invention provides the extracting methods and application of a kind of animal tendon tendon collagen, this method is to choose fresh animal tendons of beef, mutton or pork tendon, remove muscle and grease, separation obtains the tendon tissue of white, it shreds and obtains fritter tendon tissue, tissue is dissolved using low temperature acid system, obtains collagen acid solution；Low-temperature centrifugation removes undegradable tissue；Collagen supernatant obtains collagen powder by freeze-drying；The collagen powder passes through accurate weighing, is re-dissolved in acid solution, obtains certain density protein solution.The collagen passes through neutralisation treatment, can form collagen peptone, collagen film, and the functional material being variously formulated with other biological Material cladding.The advantage of the invention is that materials are convenient, cheap, preparation process is simple, stablizes, and has good biocompatibility.It can be applied to the fields such as cosmetics additive, biomaterial for medical purpose, organizational project, cell culture.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of extracting method of animal tendon tendon collagen and answer
With.
Background technique
Collagen is primarily present in the Various Tissues organ of humans and animals, is maintained in histoorgan shape and normally raw
It plays a significant role in reason function.Because it is with good biocompatibility and degradability, cell training is had been widely used for
The fields such as feeding, biology, cosmetics.In addition, the composite functional material that collagen is prepared in conjunction with other materials can be damage group
Offer bracket is provided, in conjunction with stem cell or growth factor etc. can also play the role of that function of organization is promoted to repair.
Existing commercial collagen sources mainly include Collagen type-I, Animal Skin, the collagen etc. that bovine fascia is extracted.
Collagen type-I limited source, animal skin glue ultimate constituent are more complex.Cowhells membrane tissue is more complex, and non-Collagen ingredient is difficult to remove
The defects of.
It is relatively single in order to obtain collagen component, the lower high purity collagen of immunogenicity, animal tendon tissue at
For one of optional material.Tendon tissue is fine and close, wherein main cell component-fibroblast is the main of collagen synthesis
Source, collagen component are mainly 1 Collagen Type VI.Therefore, animal tendon tissue source is sufficient, and ingredient is more single, pathogenic infection compared with
It is few, the gene of animal and people relatively, the compatibility advantages such as preferably.Therefore, the present invention extracts animal flesh by taking animal hoof as an example
Tendon collagen component, to provide new collagen material.
Summary of the invention
The object of the present invention is to provide a kind of animal tendon tendon collagen extracting method and applications.
A kind of extracting method of animal tendon tendon collagen of the present invention, specifically comprises the following steps:
(1) separating animal's tendons of beef, mutton or pork tendon: using tendon white in scissors anatomical isolation animal hoof, removal muscle, fat and
Grease and surface fascia, aseptic deionized water rinse, and obtain white tendon.
Wherein, muscle, fat, grease and whether show the removals such as fascia completely, determine the purity of tendon collagen,
It needs to remove as far as possible clean.
(2) tendon tissue is resolved into microtissue: tendon tissue passes through alcohol disinfecting, after aseptic deionized water rinses,
Tendon is shredded into micro-assembly robot with sharp scissors, sufficient deionized water is added, is siphoned away with sterile filter paper and shows oil droplet, clearly
It washes until completely removing grease.
In step (2), it is to tendon tissue disinfection, the alcohol concentration and action time are 75%-80% alcohol
Disinfection 3-5 minutes, then with sterile water wash 5 times or more.Tendon tissue shreds into (1~3) mm3Micro-assembly robot.The step be for
Pollution is reduced, tendon tissue dissolution is promoted.
(3) low temperature acid handles tendon tissue: tendon micro-assembly robot being removed water, a certain concentration acid solution is added, mixes well, sets
A couple of days is dissolved in low temperature refrigerator, during which concussion promotes dissolution.
In step (3), acid solution needed for low temperature acid handles tendon tissue is weak acid solution (such as glacial acetic acid), and volume basis contains
Amount is 0.2%-5% glacial acetic acid aqueous solution, and for low temperature range at 4-8 °, tendon micro-assembly robot and the volumn concentration are 0.2%-
The proportion of 5% glacial acetic acid aqueous solution is 2g:100ml, and dissolution time is 4-10 days, concussion daily 5 times or more.The step be in order to
Tendon tissue is promoted sufficiently to dissolve.
(4) it extracts collagen solution: solution is placed in sterile centrifugation tube, supernatant liquid, removal are collected in low-temperature and high-speed centrifugation
The undissolved tissue in bottom.
In step (4), high speed centrifugation extracts collagen solution, using centrifugal speed (10000-12000) rev/min, centrifugation
Time 10-20 minute.The step is to remove undissolved collagen tissue.
(5) collagen powder obtains: the supernatant collagen solution after centrifugation being transferred in sterile vessel, freeze-drying obtains
Collagen powder；According to amount of solution, sublimation drying is adjusted.The step is in order to which the collagen for obtaining accurate protein concentration is molten
Liquid.
(6) obtain the collagen solution of certain concentration: the collagen powder redissolution of freeze-drying needs volume basis to contain
Amount is 0.2%-2% glacial acetic acid aqueous solution, and dissolution time is 2-6 days, and can obtain concentration is that (1-15) mg/ml collagen is molten
Liquid measures solution pH value using PH detecting instrument.The step is in order to which the protein concentration and acid of quantitative collagen solution are dense
Degree.
A kind of extracting method of animal tendon tendon collagen, this kind of method are equally applicable to the hoof flesh of other animals
Tendon and tail portion tendon tissue.
A kind of application of animal tendon tendon collagen, the animal tendon tendon collagen being prepared using the above method
Protein material can be applied to following field:
(a) in cell cultivation process, table is carried out to culture carrier (culture dish/bottle, transwell, micro-fluidic chip etc.)
Face modification, promotes cell adherence and growth；
(b) cell culture, the training methods such as two and three dimensions cell；
(c) in the application of the organizational projects such as cardiac muscle, nerve, muscle, blood vessel, skin；
(d) in the cosmetologies products such as cosmetics addition, facial mask；
(e) preparation is for products such as biomaterial for medical purpose, dressing, tissue fillings；
(f) in stem cell culture and atomization；
(g) compound functional material is prepared in conjunction with other materials；
(h) food additives.
Collagen component main application fields of the present invention are related to the fields such as food, cosmetics, medicine, scientific research.
The present invention provides the extracting methods and application of a kind of animal tendon tendon collagen, the advantage of the invention is that
Materials are convenient, cheap, and preparation process is simple, stablizes, and have good biocompatibility.It can be applied to cosmetics addition
The fields such as object, biomaterial for medical purpose, organizational project, cell culture.
Detailed description of the invention
Fig. 1: photo after the separation of trotter tendon；
Fig. 2: photo after the dissolution of trotter tendon；
Fig. 3: photo is lyophilized in collagen；
Fig. 4: collagen solution photo
Fig. 5: the fourier infrared spectrogram of collagen protein powder；；
Fig. 6: collagen is used for cell culture picture；
Fig. 7: collagen is used for three-dimensional cell cultivation picture；
Fig. 8: collagen composite material prepares picture.
Specific embodiment
Embodiment 1
Prepare tendon collagen solution
The present embodiment buys trotter by taking trotter as an example, from market, is scrubbed clean with clear water, removes fur and muscle.It utilizes
White tendon in scissors anatomical isolation trotter, removal muscle, fat and grease and surface fascia, aseptic deionized water are rinsed, are obtained
Obtain white tendon (Fig. 1).Tendon tissue passes through alcohol disinfecting 3-5 minute, aseptic deionized water flushing 3 times or more, with sharp
Tendon is shredded into 3-5 millimeters of micro-assembly robots (Fig. 2) by scissors, and sufficient deionized water is added and rinses 3-5 flushing, with sterile filter
Paper, which siphons away, shows oil droplet, and cleaning is until completely remove grease.Dose volume percentage composition is 0.2% glacial acetic acid aqueous solution, filtering
4 degree of degerming spare.The tendon tissue shredded is added in glacial acetic acid according to 0.5% mass volume ratio, abundant suspended tissue sets
It is dissolved 10 days in 4 degree of refrigerators, concussion daily 5 times or more, promotes tendon tissue dissolution.Dissolved gum original solution is transferred to 50 millis
Rise in centrifuge tube, 4 degree high speed centrifugation 15 minutes, revolving speed is per minute 1.5 ten thousand.Upper solution is collected, removal bottom is undissolved
Tissue.Collagen solution supernatant will be obtained to be transferred in 10 centimetres of sterile culture dishes, be freeze-dried 20-30 hours, obtain tendon
Collagen powder (Fig. 3).Quantitative under aseptic condition, being redissolved in volumn concentration is 4 degree of ice in 0.2% glacial acetic acid aqueous solution
Case dissolves 6 days, and can obtain concentration is (1-15) mg/ml collagen solution (Fig. 4).
Fig. 1 is that separation obtains animal hoof tendon, is about 10 centimetres.Fig. 2 is that the tendon tissue shredded is placed in sterile water.Figure
3 be the collagen powder after collagen solution freeze-drying.Fig. 4 is that collagen powder is redissolved in formation flat-white emulsion in acid solution
Liquid.Fig. 5 is the fourier infrared spectrogram of collagen protein powder.As shown in Figure 5, the present invention infrared figure of tendon collagen obtained
It is similar that spectrum mainly goes out peak position to type i collagen.3288cm1Neighbouring N-H stretching vibration, 1627cm1It is nearby that the C-O of amide I band stretches
Contracting vibration, 1525cm1It is nearby the N-H bending vibration of amide II band, 1444~1235cm1Neighbouring absorption peak shows collagen
The integrality of 3 strands of helical structures of albumen.It can be seen that tendon collagen main component is type i collagen.
The present embodiment has attempted 0.2%, 0.4%, 1%, 2%, 4%, and the acetic acid solution of 5% volume ratio is to tendon tissue
Degradation speed, the higher degradation time of acetic acid concentration is shorter as the result is shown, and acquisition is that collagen is more.
Embodiment 2
The application of collagen
(1) collagen is used for cell culture:
Collagen is diluted to the solution of (80-200) ug/ml with 0.2% glacial acetic acid, is coated with glass and plastic culture
Ware, 4 spend night, 37 degree of solidifications 4 hours or more.Or 60-70 degree oven is dried 4 hours or more.It is cleaned with sterile PBS, in
With extra acid solution.Addition Human umbilical vein endothelial cells and primary glomerular endothelial cells suspension, stationary culture 3 days (Fig. 6).
Fig. 6 is to cultivate Human umbilical vein endothelial cells and rat primary glomerulus endothelium after collagen is coated with glass culture dish
Cell.Fig. 6 A is HE dyeing, and Fig. 6 B is CD31 immunofluorescence dyeing.Cellular morphology is typical paving stone sample.
(2) three-dimensional cell cultivation
10mg/ml collagen is neutralized into 6mg/ml solution with PBS, tumour cell U87 is added, is prepared into suspension, adds
Enter into sodium alginate silk, carries out dimensional culture (Fig. 7).Fig. 7 shows the U87 cell mixed with collagen solution in three-dimensional strip
Well-grown under part.
Embodiment 3
The preparation of collagen composite material
Collagen is mixed with chitosan, according to Conventional porous timbering material preparation method, can prepare composite material, and
For Three-dimensional cell culture.Fig. 8 is collagen/chitosan three-dimensional stent material.
Above-described embodiment only technical concept and feature to illustrate the invention, its object is to allow person skilled in the art
Member cans understand the content of the present invention and implement it accordingly, and it is not intended to limit the scope of the present invention.It is all according to the present invention
The equivalent change or modification that Spirit Essence is done, should be covered by the protection scope of the present invention.
Claims (8)
1. a kind of extracting method of animal tendon tendon collagen, it is characterised in that follow the steps below:
(1) tendon white in scissors anatomical isolation animal hoof, removal muscle, fat and grease separating animal's tendons of beef, mutton or pork tendon: are utilized
With surface fascia, aseptic deionized water is rinsed, and obtains white tendon；
(2) tendon tissue is resolved into microtissue: tendon tissue passes through alcohol disinfecting and uses cutting edge of a knife or a sword after aseptic deionized water rinses
Tendon is shredded into micro-assembly robot by the scissors of benefit, and sufficient deionized water is added, is siphoned away with sterile filter paper and shows oil droplet, and cleaning is straight
To completely removing grease；
(3) low temperature acid handles tendon tissue: tendon micro-assembly robot being removed water, a certain concentration acid solution is added, mixes well, is placed in low
Temperature refrigerator dissolves a couple of days, and during which concussion promotes dissolution；
(4) it extracts collagen solution: solution is placed in sterile centrifugation tube, low-temperature and high-speed centrifugation collects supernatant liquid, removes bottom
Undissolved tissue；
(5) freeze-drying obtains tendon collagen powder: the upper layer collagen solution after centrifugation is placed in sterile petri dish, it is low
Temperature freeze-drying, obtains tendon collagen powder；
(6) quantitative collagen solution concentration: under aseptic condition, accurate weighing collagen powder is re-dissolved in sterile low
In warm acid solution, dissolution in low temperature is complete, obtains certain density collagen solution.
2. according to a kind of extracting method of animal tendon tendon collagen described in claim 1, it is characterised in that: step (2)
In, to the alcohol disinfecting, specially use 75%-80% alcohol disinfecting 3-5 minutes, the sterile water wash 5 times or more.
3. according to a kind of extracting method of animal tendon tendon collagen described in claim 1, it is characterised in that: step (2)
In, tendon tissue shreds into 1~3mm3Micro-assembly robot.
4. according to a kind of extracting method of animal tendon tendon collagen described in claim 1, it is characterised in that: step (3)
In, a certain concentration acid solution is weak acid solution；Specifically: volumn concentration is the glacial acetic acid aqueous solution of 0.2%-5%,
Tendon micro-assembly robot and the volumn concentration are that the proportion of 0.2%-5% glacial acetic acid aqueous solution is 2g:100ml；The low temperature
It is 4-8 °；Dissolution time is 4-10 days, concussion daily 5 times or more.
5. according to a kind of extracting method of animal tendon tendon collagen described in claim 1, it is characterised in that: step (4)
In, the high speed centrifugation, using 10000-12000 revs/min of centrifugal speed, centrifugation time 10-20 minutes.
6. according to a kind of extracting method of animal tendon tendon collagen described in claim 1, it is characterised in that: step (6)
In, acid solution at low temperature is that volumn concentration is 0.2%-2% glacial acetic acid aqueous solution, and dissolution time is 2-6 days, can be obtained dense
Degree is (1-15) mg/ml collagen solution.
7. according to a kind of extracting method of animal tendon tendon collagen described in claim 1, it is characterised in that the animal
For the hoof tendon tissue of pig, ox and other larger animals.
8. a kind of application of animal tendon tendon collagen, which is characterized in that the animal hoof being prepared using the above method
Muscle tendon collagen material, the material are applied to following field:
(a) in cell cultivation process, culture carrier is surface modified, promotes cell adherence and growth；The culture carrier
For culture dish/bottle, transwell, micro-fluidic chip and other carriers；
(b) cell 3D is cultivated；
(c) in the application of the organizational projects such as cardiac muscle, nerve, muscle, blood vessel, skin；
(d) in the cosmetologies products such as cosmetics addition, facial mask；
(e) preparation is for products such as biomaterial for medical purpose, dressing, tissue fillings；
(f) in stem cell culture and atomization；
(g) compound functional material is prepared in conjunction with other materials；
(h) food additives.
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| CN112826757A (en) * | 2020-12-16 | 2021-05-25 | 中新国际联合研究院 | Antibacterial and anti-inflammatory composite collagen essence and preparation method and application thereof |
| RU2791324C1 (en) * | 2021-12-27 | 2023-03-07 | федеральное государственное бюджетное образовательное учреждение высшего образования "Алтайский государственный университет" | Process for obtaining collagen gel for use in medicine and cosmetology |
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|---|---|---|---|---|
| CN112826757A (en) * | 2020-12-16 | 2021-05-25 | 中新国际联合研究院 | Antibacterial and anti-inflammatory composite collagen essence and preparation method and application thereof |
| RU2791324C1 (en) * | 2021-12-27 | 2023-03-07 | федеральное государственное бюджетное образовательное учреждение высшего образования "Алтайский государственный университет" | Process for obtaining collagen gel for use in medicine and cosmetology |
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