CN103333241A - Technology of preparing acid-soluble bovine tendo calcaneus collagen by freeze thawing-ultrasonic method - Google Patents
Technology of preparing acid-soluble bovine tendo calcaneus collagen by freeze thawing-ultrasonic method Download PDFInfo
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- CN103333241A CN103333241A CN2013102956674A CN201310295667A CN103333241A CN 103333241 A CN103333241 A CN 103333241A CN 2013102956674 A CN2013102956674 A CN 2013102956674A CN 201310295667 A CN201310295667 A CN 201310295667A CN 103333241 A CN103333241 A CN 103333241A
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Abstract
The invention relates to a technology of preparing acid-soluble bovine tendo calcaneus collagen by a freeze thawing-ultrasonic method. The technology comprises the specific steps of: (1) bovine tendo calcaneus pretreatment: removing impurities from fresh bovine tendo calcaneus, soaking with normal saline, washing with pure water, freezing, slicing and airing, (2) swelling: putting and soaking aired bovine tendo calcaneus slices in an acetic acid solution for swelling, (3) beating: adding pure water into a swelled bovine tendo calcaneus suspension, performing mechanical beating to obtain crude slurry, (4) freeze thawing-ultrasonic: freezing the beaten bovine tendo calcaneus crude slurry, then performing ultrasonic treatment, repeating the freeze thawing-ultrasonic process to obtain a crude collagen extract, and (5) purification: performing filtering, centrifuging, salt precipitation, dialysis and freeze drying on the crude collagen extract to obtain the collagen product. The extraction method of the bovine tendo calcaneus collagen is simple and convenient and low in cost, the extraction rate of the collagen is increased without using enzyme, the purity of the obtained collagen is high, and a natural structure of the collagen is reserved. The prepared collagen can be applied to industries of medicine, cosmetics and the like.
Description
Technical field
The present invention relates to the technology that a kind of freeze thawing-ultrasonic method prepares sour molten ox heel string collagen, relate in particular to a kind of preparation technology of non-Enzymatic Extraction natural collagen.Belong to collagen extractive technique field in the organism.
Background technology
In vertebrate skin, bone, tendon, contain a large amount of collagen.Its type is varied, and content is the abundantest and to use more be type i collagen.Ox heel string source is abundant, and the content of type i collagen is very high, is to extract the good raw material sources of type i collagen.The type i collagen molecule is coiled to form unique triple helix structure by three peptide chains, contains the Gly-X-Y aminoacid sequence of a large amount of repetitions in every chain, and wherein proline(Pro) and hydroxyproline content are higher.Contain a large amount of hydrogen bonds in the tropocollagen molecule, be full of in the tropocollagen molecule chain and interchain.These hydrogen bonds not only can be stablized the triple helix structure of collagen, have also given collagen very high water binding ability, make collagen be widely used in fields such as makeup or food as wetting Agent for Printing Inks.The tropocollagen molecule polymer fiber has the favorable mechanical performance, adds that the biocompatibility of itself has been given its application prospect wide on biomaterial jointly.Collagen also can promote platelet aggregation, and it can obtain amino acid as a kind of cell matrix after degraded, promotes the growth of cell for cell provides nutrient, and the Chang Zuowei hemostatic material is applied to medical field.Obtain gelatin after the collagen process hydrolysis sex change, gelatin and natural collagen all may be used on playing moisture-keeping function in the makeup, but unmodified natural collagen has than the better water retention of gelatin and cell adhesion effect.
Collagen has multiple extracting method.Distinguish according to extracting the effective constituent that collagen uses, can be divided into alkaline process extractions, neutral sulfity process extraction, Enzymatic Extraction, acid system extraction, and being used in combination of these methods.The use of alkali can cause the collagen peptide bond hydrolysis, and the amino acid meeting of hydroxyl and sulfydryl is destroyed and produce the racemization phenomenon, so alkaline process is not suitable for extracting the collagen that needs to keep native configurations.Owing to contain less in the great majority tissues even do not have the neutral salt procollagen, it is lower therefore to utilize the neutral salt extraction method to prepare the yield of collagen.Enzymatic Extraction collagen can keep the triple helix structure of tropocollagen molecule, and productive rate is higher, is the extracting method of at present the most frequently used natural collagen.Disclose a kind of preparation method of collagen protein such as Chinese patent CN200810156070.0, its raw material sources are beef tendon, extract the collagen that obtains and have kept the triple helix structure, and extracting mode is the proteolysis enzyme extraction.Though Enzymatic Extraction natural collagen yield is higher, enzyme and collagen belong to protein together, and the later stage separation difficulty increases cost, if do not separate, the existence of enzyme will influence purity and the range of application of collagen.
Acid can make the collagen swelling, and experimental observation is to the ox heel string behind acid-swellable, and its weight can increase to tens of times of dry weight.Directly soak ox heel string extraction collagen wherein with organic acid such as acetic acid, extraction yield is very low, thereby acid system usually is used in combination to extract collagen with enzyme process.If can under the prerequisite that does not adopt enzyme, extract natural collagen with acid system, and improve the extraction yield of collagen, will have great significance to the widespread use of collagen.Hyperacoustic use can make collegen filament lax, cooperates the multigelation method can improve the efficient of acid extraction collagen.
Summary of the invention
The objective of the invention is: propose a kind of preparation method who keeps the ox heel string collagen of natural triple helix structure, especially a kind of method of non-enzyme extraction of taking is to keep the highly purified preparation method of collagen.
Technical solution of the present invention is: the preparation method of collagen, step is as follows: (1) pre-treatment: after fresh ox heel string is rejected manadesma and fat, be cut into small pieces and soak with physiological saline, after pure water rinsing, freezing 5h-20h under-20 ℃ of-0 ℃ of conditions, and thinly slice, dry, obtain ox heel string thin slice; (2) swelling: the ox heel string thin slice after will drying places acetic acid solution to soak swelling 20h-40h by mass volume ratio (ox heel string thin slice: acetic acid solution) be 1:(100-300); (3) making beating: press mass volume ratio (ox heel string thin slice: pure water) be 1:(100-500) in the ox heel string suspension liquid after the swelling and add pure water, machinery is pulled an oar, and obtains thick slurries; (4) freeze thawing-ultrasonic: with thick slurries freezing 5h-20h under-60 ℃ of-0 ℃ of conditions, after pressing mass volume ratio (ox heel string thin slice: pure water) be 1:(10-100) adding pure water, under the ice-water bath condition, carry out ultrasonic 1h-3h, repeat freeze thawing-ultrasonic procedure 1-5 time, obtain the collagen crude extract; (5) purifying: with gained collagen crude extract filter, centrifugal, with sodium chloride solution saltout the back under 4 ℃ of conditions with the pure water dialysis, till the water for dialysis stable conductivity, lyophilize namely gets the collagen product.
Wherein, the acetic acid solution described in the step (2) is the acetic acid solution of volume fraction 10%-80%, and swelling time is optimized for 20h-40h.
Thick slurries after freezing described in the step (4) namely carry out supersound process without thawing.
But advantage inductive generalization of the present invention is as follows.
(1) improved the extraction yield that common acid system extracts collagen, the process of extraction is easy, and the reagent type of use is few, does not add enzyme, and cost is low.
(2) the collagen product that the present invention is made carries out infrared, DSC(differential scanning calorimeter) and detection such as Zeta potential, the result shows that the collagen that the present invention makes has kept natural triple helix structure, and purity is higher.
(3) guaranteed that the collagen of natural structure is all good than denatured collagen in the performance of aspects such as machinery, water conservation, and because leaching process does not add enzyme, the prepared collagen of the present invention can be applied even more extensively in medicine, cosmetic field.
Embodiment
Embodiment one1. pre-treatment: buy fresh ox heel string, with manadesma, the fat on the stainless pocket knife rejecting tendon.The ox heel string of removing manadesma and fat is cut into fritter long about 1cm.Soak 18h with physiological saline, change physiological saline and soak about 2h again.Take out ox heel string fritter, comparatively clarify with pure water rinsing to washing fluid.Ox heel string fritter after the flushing is wrapped with preservative film after with the stainless steel filtering net drainage, and the refrigerator and cooled that is placed on-20 ℃ is frozen 18h.Take out the ox heel string fritter after freezing, be cut into thickness less than the thin slice of 0.5mm.Ox heel string sheet is disperseed to be layered on the preservative film, be positioned on the super clean bench and ventilate, dry.Collect the tendon sheet that dries after three days, stand-by;
2. swelling: in the beaker of 500mL, add the 300mL volume fraction and be 20% acetic acid solution and 3g ox heel string sheet.Beaker is sealed with preservative film, placed 24h at ambient temperature;
3. making beating: add pure water 600mL in the ox heel string suspension liquid after the swelling, with the refiner making beating, obtain thick slurries;
4. freeze thawing-ultrasonic: with thick slurries freezing 8h under-20 ℃ of conditions, to wherein adding the 10mL pure water, under the ice-water bath condition, carry out ultrasonic 3h, repeat freeze thawing-ultrasonic procedure 2 times, obtain the collagen crude extract;
5. purifying: with the collagen crude extract filter, centrifugal.Centrifugal employing high speed freezing centrifuge, rotating speed are 2500r/min, and temperature is 8 ° of C, and centrifugation time is 40min.Pour out supernatant liquid after centrifugal, the sodium chloride solution that adds 2mol/L is saltoutd.The collagen that obtains saltouing is packed in the dialysis tubing, with pure water dialyse (dialysis tubing with preceding use boiling pure water more than half hour).Dialysis cassette is put into refrigerator for cold, changes water every day three or four times, the specific conductivity of at every turn changing water Pretesting water for dialysis, and it is stable to repeat to carry out dialysis dialysis back electrical conductivity of water.After having dialysed, with the stored refrigerated in the beaker of packing into of the collagen in the dialysis tubing.Heel string fragment after the filtration carries out second extraction through extraction of the present invention and purification step again.
Embodiment two1. pre-treatment: buy fresh ox heel string, with manadesma, the fat on the stainless pocket knife rejecting tendon.The ox heel string of removing manadesma and fat is cut into fritter long about 1cm.Soak 24h with physiological saline.Take out ox heel string fritter, comparatively clarify with pure water rinsing to washing fluid.Ox heel string fritter after the flushing is wrapped with preservative film after with the stainless steel filtering net drainage, and the refrigerator and cooled that is placed on-20 ℃ is frozen 12h.Take out the ox heel string fritter after freezing, be cut into thickness less than the thin slice of 0.5mm.Ox heel string sheet is disperseed to be layered on the preservative film, be positioned on the super clean bench and ventilate, dry.Collect the tendon sheet that dries after three days, stand-by;
2. swelling: in the beaker of 500mL, add the 400mL volume fraction and be 30% acetic acid solution and 3g ox heel string sheet.Beaker is sealed with preservative film, placed 24h at ambient temperature;
3. making beating: add pure water 600mL in the ox heel string suspension liquid after the swelling, with the refiner making beating, obtain thick slurries;
4. freeze thawing-ultrasonic: with thick slurries freezing 15h under-30 ℃ of conditions, add pure water 20mL, under the ice-water bath condition, carry out ultrasonic 2h, repeat freeze thawing-ultrasonic procedure 3 times, obtain the collagen crude extract;
5. purifying: with the collagen crude extract filter, centrifugal.Centrifugal employing high speed freezing centrifuge, rotating speed are 2500r/min, and temperature is 8 ° of C, and centrifugation time is 40min.Pour out supernatant liquid after centrifugal, the sodium chloride solution that adds 2mol/L is saltoutd.The collagen that obtains saltouing is packed in the dialysis tubing, with pure water dialyse (dialysis tubing with preceding use boiling pure water more than half hour).Dialysis cassette is put into refrigerator for cold, changes water every day three or four times, the specific conductivity of at every turn changing water Pretesting water for dialysis, and it is stable to repeat to carry out dialysis dialysis back electrical conductivity of water.After having dialysed, with the stored refrigerated in the beaker of packing into of the collagen in the dialysis tubing.Heel string fragment after the filtration carries out second extraction through extraction of the present invention and purification step again.
Embodiment three1. pre-treatment: buy fresh ox heel string, with manadesma, the fat on the stainless pocket knife rejecting tendon.The ox heel string of removing manadesma and fat is cut into fritter long about 1cm.Soak 24h with physiological saline, change physiological saline and soak about 2h again.Take out ox heel string fritter, comparatively clarify with pure water rinsing to washing fluid.Ox heel string fritter after the flushing is wrapped with preservative film after with the stainless steel filtering net drainage, and the refrigerator and cooled that is placed on-20 ℃ is frozen 18h.Take out the ox heel string fritter after freezing, be cut into thickness less than the thin slice of 0.5mm.Ox heel string sheet is disperseed to be layered on the preservative film, be positioned on the super clean bench and ventilate, dry.Collect the tendon sheet that dries after three days, stand-by;
2. swelling: in the beaker of 500mL, add the 500mL volume fraction and be 40% acetic acid solution and 3g ox heel string sheet.Beaker is sealed with preservative film, placed 24h at ambient temperature;
3. making beating: add pure water 800mL in the ox heel string suspension liquid after the swelling, with the refiner making beating, obtain thick slurries;
4. freeze thawing-ultrasonic: with thick slurries freezing 20h under-40 ℃ of conditions, add pure water 20mL, under the ice-water bath condition, carry out ultrasonic 1h, repeat freeze thawing-ultrasonic procedure 4 times, obtain the collagen crude extract;
5. purifying: with the collagen crude extract filter, centrifugal.Centrifugal employing high speed freezing centrifuge, rotating speed are 2500r/min, and temperature is 8 ° of C, and centrifugation time is 40min.Pour out supernatant liquid after centrifugal, the sodium chloride solution that adds 2mol/L is saltoutd.The collagen that obtains saltouing is packed in the dialysis tubing, with pure water dialyse (dialysis tubing with preceding use boiling pure water more than half hour).Dialysis cassette is put into refrigerator for cold, changes water every day three or four times, the specific conductivity of at every turn changing water Pretesting water for dialysis, and it is stable to repeat to carry out dialysis dialysis back electrical conductivity of water.After having dialysed, with the stored refrigerated in the beaker of packing into of the collagen in the dialysis tubing.Heel string fragment after the filtration carries out second extraction through extraction of the present invention and purification step again.
Claims (3)
1. a freeze thawing-ultrasonic method prepares the technology of sour molten ox heel string collagen, it is characterized in that, comprises the following steps:
(1) pre-treatment: behind fresh ox heel string rejecting manadesma and fat, be cut into small pieces and soak with physiological saline, after pure water rinsing, freezing 5h-20h under-20 ℃ of-0 ℃ of conditions, and thinly slice, dry, obtain ox heel string thin slice;
(2) swelling: the ox heel string thin slice after will drying is by mass volume ratio (ox heel string thin slice: 1:(100-300 acetic acid solution)) place acetic acid solution to soak swelling 20h-40h;
(3) making beating: (ox heel string thin slice: 1:(100-500 pure water)) add pure water, machinery is pulled an oar, and obtains thick slurries press mass volume ratio in the ox heel string suspension liquid after the swelling;
(4) freeze thawing-ultrasonic: with thick slurries freezing 5h-20h under-60 ℃ of-0 ℃ of conditions, press mass volume ratio (ox heel string thin slice: 1:(10-100 pure water)) behind the adding pure water, under the ice-water bath condition, carry out ultrasonic 1h-3h, repeat freeze thawing-ultrasonic procedure 1-5 time, obtain the collagen crude extract;
(5) purifying: with gained collagen crude extract filter, centrifugal, with sodium chloride solution saltout the back under 4 ℃ of conditions with the pure water dialysis, till the water for dialysis stable conductivity, lyophilize namely gets the collagen product.
2. prepare the technology of sour molten ox heel string collagen according to the described freeze thawing-ultrasonic method of claim 1, it is characterized in that, the acetic acid solution described in the step (2) is the acetic acid solution of volume fraction 10%-80%.
3. prepare the technology of sour molten ox heel string collagen according to the described freeze thawing-ultrasonic method of claim 1, it is characterized in that, the thick slurries behind freezing described in the step (4) namely carry out supersound process without thawing.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105131109A (en) * | 2015-09-30 | 2015-12-09 | 陕西艾尔肤组织工程有限公司 | Collagen extracting method |
| CN107691977A (en) * | 2017-10-10 | 2018-02-16 | 广西金诚双丰农牧科技有限公司 | The preparation method of bamboo rat jerky with strengthening spleen and nourishing stomach effect |
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| CN1915437A (en) * | 2005-08-15 | 2007-02-21 | 上海其胜生物制剂有限公司 | Technique for preparing sponge produced from collagen |
Non-Patent Citations (2)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105131109A (en) * | 2015-09-30 | 2015-12-09 | 陕西艾尔肤组织工程有限公司 | Collagen extracting method |
| CN107691977A (en) * | 2017-10-10 | 2018-02-16 | 广西金诚双丰农牧科技有限公司 | The preparation method of bamboo rat jerky with strengthening spleen and nourishing stomach effect |
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