CN102952188A - Method for preparing high-purity collagens through using fish skins as raw material - Google Patents
Method for preparing high-purity collagens through using fish skins as raw material Download PDFInfo
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Abstract
The invention provides a method for preparing high-purity collagens through using fish skins as a raw material. The method sequentially comprises the following steps: 1, crushing the fish skins; 2, degreasing; 3, dissolving out non-collagen proteins; 4, cleaning; 5, dissolving out collagens; 6, concentrating a collagen solution; and 7, drying the collagens. The non-collagen proteins in the fish skins are first dissolved out under a temperature lower than a collagen denaturation temperature, the temperature is controlled in a collagen denaturation temperature range, and the dissolving time is shorter than a time required by the collagen denaturation, so the collagens having a low or zero hydrolysis degree can be obtained. The collagens have a natural structure, and have the advantages of high molecular weight, high purity and the like.
Description
[technical field]
The present invention relates to the method that a kind of collagen protein extracts, especially a kind of method for preparing high purity collagen take fish-skin as raw material.
[background technology]
Collagen protein is a kind of fibrous protein that are comprised of with the right-handed helix form 3 polypeptide chains, almost is present in institute in a organized way as the important composition of extracellular matrix, is the rich in protein of animal body intensive amount.That it is used in fields such as functional food, healthcare products, biomaterial, organizational project and bio-medical materials is very extensive because having the characteristics such as reduced immunogenicity, good biocompatibility and biodegradable for collagen protein.Collagen protein commonly used mainly is mammiferous skin and osseous tissue and fish-skin or fish phosphorus etc.Application and its molecular weight ranges and the purity of collagen protein in a lot of fields is closely related, the collagen protein of using in the fields such as biology and medical science mainly is high molecular and highly purified collagen protein, the collagen protein that is used for the aspects such as food typing at field of food mainly is the high molecular collagen protein, and therefore preparing high molecular and high-purity collagen protein is the key of expanding its range of application.Because collagen protein is difficult at normal temperatures water-soluble solution and is difficult to the interior bio-enzyme degradation of body, therefore prepare collagen protein and often use the combination of acid system, alkaline process, dense salt method, enzyme process, heat drop solution or these methods with the rear stripping of its degraded.Ceng Shaokui etc. have reported and have utilized acid solution to extract collagen protein from the tilapia fish scale and use enzyme process to prepare method (Shanghai Ocean University's journal, 2009,18 (5): 599-603) of collagen polypeptide; Liu Yan etc. have studied method (aquatic science, 2010,29 (4): 221-224) of using enzyme process to extract collagen protein from 4 kinds of freshwater fish scales; Once Rong had reported that hydrochloric acid decalcification, enzyme be combined method (biological processing, 2008,6 (5): 27-30) of extracting collagen protein with citric acid; Chen Shengjun etc. have studied technique (aquatic science, 2004,23 (6): 45-46) that alkaline process prepares collagen protein; In addition, use separately the heat drop solution also can be so that collagen protein stripping (National University of the Inner Mongol's journal, 2010,25 (5): 525-529).Patent (application number 201010513190.9) has been announced a kind of recombining process that belongs to acid extraction-aspartic protease enzymolysis, and patent (application number 201010192216.4) has announced a kind of use column chromatography technology and the binding film separating technology prepares the collagen protein method; Although it is existing many to extract the research of collagen protein, there are many defectives in traditional collagen protein extracting method, mainly comprises:
(1) the collagen molecules weight range is wide, because these traditional methods mainly are wide with improving its water-soluble collagen molecules weight range of preparing of causing after the collagen protein degraded, usually is hundreds of to twenties ten thousand dalton.
(2) lipidated protein is not high, and traditional method is blended into collagen solution after preparing the albumen stripping of non-collagen class in the collagen protein process, causes the collagen protein purity prepared lower.
(3) the collagen protein sequence changes, and exists a certain amount of l-asparagine and glutamine residue, traditional method to prepare the amino-acid residue generation deacylated tRNA amine phenomenon of these amide containings in the collagen protein process in the aminoacid sequence of collagen protein.
Collagen property and its animal-origin are close, the collagen protein in Mammals source has preferably thermostability, and the collagen protein thermostability of fish is lower than the collagen protein in Mammals source, the collagen protein that uses traditional extracting method to extract from fish-skin seriously causes its molecular-weight average not high owing to degrading, therefore, extraction high molecular and high purity collagen are more difficult take fish-skin as raw material.
[summary of the invention]
The object of the invention is to provide a kind of method for preparing high molecular and high purity collagen take fish-skin as raw material.
Therefore, the invention provides a kind of method for preparing high purity collagen take fish-skin as raw material, it is characterized in that, the method may further comprise the steps successively: (1) fish-skin is pulverized; (2) degreasing; (3) non-SUB1095 stripping; (4) clean; (5) collagen protein stripping; (6) collagen solution is concentrated; (7) collagen protein is dry.
The thermostability of non-SUB1095 is lower than the thermostability of collagen protein in the fish-skin, is being lower than under the collagen protein denaturation temperature condition, adds the at first stripping of protein that acidic solution can make non-collagen class in the fish-skin; The temperature of solution in the leaching process is controlled at collagen protein variable temperature occurs, can make collagen protein stripping in the fish-skin, thereby can prepare the collagen protein with higher molecular weight range, simultaneously, before extracting collagen protein, with the protein stripping of non-collagen class, provide the purity of the collagen protein of finally preparing.
[description of drawings] Fig. 1 is the process flow sheet that extracts high-purity collagen protein take fish-skin as raw material.
[embodiment]
The invention provides a kind of method for preparing high purity collagen take fish-skin as raw material, it is characterized in that, the method may further comprise the steps successively: (1) fish-skin is pulverized; (2) degreasing; (3) non-SUB1095 stripping; (4) clean; (5) collagen protein stripping; (6) collagen solution is concentrated; (7) collagen protein is dry.
In the present invention, described fish-skin can be untreated dried fish-skin or wet fish-skin, dried fish-skin or wet fish-skin after perhaps pulverizing; Also can be the fish-skin through degreasing and/or deliming processing.
In the present invention, described step (3), (4) and (5) can be carried out in the container of solid-liquid by continuous separate any, but a kind of preferred embodiment in, all carry out in extractor described step (3), (4) and (5), and described extractor has the filtration unit of holding back fish-skin.
Step (1) fish-skin is pulverized and (2) degreasing all can be carried out according to prior art.A kind of preferred embodiment in, described degreasing is to inject continuously grease-removing agent in extractor, flows out in extractor after grease-removing agent contacts with fish-skin, and the grease-removing agent add-on is 10-20 times of hygrometric state fish-skin weight, injection length is controlled at 18-32 hour, and temperature is controlled at below 18 ℃.Grease-removing agent can be the isopropanol water solution of 5～30 volume % or the n-butanol aqueous solution of 5～30 volume %.
In described step (3), the stripping of non-collagen proteinoid mainly is by controlling the temperature of this step, the temperature that common this step of control is carried out is lower than the critical temperature of collagen of fish skin generation sex change, make the stripping from fish-skin of non-collagen proteinoid, and collagen protein still is retained in the fish-skin, remove non-collagen proteinoid thereby whereby collagen protein is separated with non-collagen proteinoid, can be water or aqueous acid for the medium that dissolves non-collagen proteinoid.
A kind of preferred embodiment in, in described step (3), fish-skin is contacted with acidic aqueous solution, and controls the critical temperature that described Contact Temperature is lower than described collagen of fish skin generation sex change, make the noncollagen protein stripping in described acidic aqueous solution.
In another preferred embodiment, in described step (3), described noncollagen protein stripping step is continuous mode, and the injection of described acidic aqueous solution can be injected continuously, and the aqueous solution that is dissolved with noncollagen protein flows out continuously.A kind of preferred embodiment in, described non-SUB1095 stripping step is continuous, the condition that adopts comprises: inject continuously acidic aqueous solution in extractor, after contacting with the hygrometric state fish-skin, described acidic aqueous solution flows out from extractor, discharge rate equates with the rate of injection of described acidic aqueous solution, temperature is controlled at 15～40 ℃ in the extractor, the acid concentration of described acidic aqueous solution is 0.01～0.05mol/L, described acidic aqueous solution is 10: 1～20: 1 with the ratio of the weight of described hygrometric state fish-skin, and the time of injecting continuously acidic aqueous solution is controlled in 6～12 hours scopes.
In another preferred embodiment, described acidic aqueous solution is the aqueous solution of acetic acid, citric acid or hydrochloric acid.
In described step (3), when when injecting described acidic aqueous solution continuously, the speed of injecting continuously described acidic aqueous solution can be identical or different with the speed that the aqueous solution that is dissolved with noncollagen protein flows out continuously, but for the aqueous solution that is dissolved with noncollagen protein can be flowed out continuously, the speed of injecting continuously described acidic aqueous solution should be more than or equal to the continuous speed that flows out of the aqueous solution that is dissolved with noncollagen protein.But a kind of preferred embodiment in, the speed of injecting continuously described acidic aqueous solution equates with the speed that the aqueous solution that is dissolved with noncollagen protein flows out continuously, so that noncollagen protein one stripping separates with the fish-skin that contains collagen protein with regard at once outflow.
Described cleaning step (4) is continuous mode in the present invention, the condition that adopts comprises: continuous injected water, after contacting, fish-skin after making described water and processing through step (3) flows out, discharge rate can be identical or different with the speed of injecting continuously described water, but for scavenging solution can be flowed out continuously, the speed of injecting continuously described water should be more than or equal to discharge rate.A kind of preferred embodiment in, described cleaning step is continuous, the condition that described method adopts comprises: inject continuously clear water in described extractor, after contacting with the hygrometric state fish-skin, described clear water flows out from described extractor, discharge rate equates with the rate of injection of described clear water, temperature is controlled at 10～30 ℃ in the described extractor, and described clear water is 6: 1～8: 1 with the ratio of the weight of described hygrometric state fish-skin, and the time of injecting continuously described clear water was controlled in 1 hour.
In step of the present invention (5), the extraction of collagen protein mainly is adding speed and the discharge rate by the temperature of controlling this step and clear water.Usually the temperature of this step is controlled at the critical temperature of collagen of fish skin generation thermally denature, makes collagen protein stripping from fish-skin under the thermally denature condition, and temperature reduced after the collagen solution of stripping flowed out, and the collagen protein of sex change recovers its native state gradually.A kind of preferred embodiment in, described collagenic protein stripping step is continuous, the condition that adopts comprises: implantation temperature is 55～95 ℃ clear water continuously in the described extractor, described clear water is with after described hygrometric state fish-skin contacts, collagen protein stripping from described hygrometric state fish-skin is flowed out to clear water and in described extractor, discharge rate equates with the rate of injection of described clear water, collect effluent liquid, temperature is controlled at 55～95 ℃ in the described extractor, described clear water is 10: 1～30: 1 with the ratio of the weight of the described fish-skin of described hygrometric state, and the time of injecting continuously described 55～95 ℃ of clear water is controlled at 3～10 hours.
In step of the present invention (5), the speed of injecting continuously clear water can be identical or different with the speed that the aqueous solution that is dissolved with collagen protein flows out continuously, but for the aqueous solution that is dissolved with collagen protein can be flowed out continuously, the speed of injecting continuously described clear water should be more than or equal to the continuous speed that flows out of the aqueous solution that is dissolved with collagen protein.A kind of preferred embodiment in, the speed of injecting continuously described clear water equates with the speed that the aqueous solution that is dissolved with collagen protein flows out continuously, avoids the further thermally denature of collagen protein of stripping so that flow out at once after the collagen protein stripping.
In a kind of most preferred embodiment, method of the present invention comprises:
(1) fish-skin is pulverized: through water soaking, the elimination aqueous solution adopts pulverizer that fish-skin is ground into bulk, is loaded in the extractor with fish-skin, and extractor has the filtration unit of holding back fish-skin.
(2) degreasing: inject continuously grease-removing agent in extractor, flow out in extractor after grease-removing agent contacts with fish-skin, the grease-removing agent add-on is 10-20 times of hygrometric state fish-skin weight, and injection length is controlled at 18-32 hour, and temperature is controlled at below 18 ℃.Grease-removing agent can be the isopropanol water solution of 5～30 volume % or the n-butanol aqueous solution of 5～30 volume %.
(3) non-SUB1095 stripping: inject continuously acidic aqueous solution in extractor, flow out from extractor after acidic aqueous solution contacts with the hygrometric state fish-skin, the rate of injection of control discharge rate and acidic aqueous solution equates.Temperature is controlled at 15～40 ℃ in the extractor.The acid concentration of described acidic aqueous solution is 0.01～0.05mol/L, and described acidic aqueous solution is 10: 1～20: 1 with the ratio of the weight of the described fish-skin of hygrometric state, and the time of injecting continuously acidic aqueous solution is controlled in 6～12 hours scopes.
(4) clean: inject continuously clear water in extractor, flow out from extractor after clear water contacts with fish-skin, the rate of injection of control discharge rate and clear water equates; Temperature is controlled at 15～30 ℃ in the extractor, and clear water is 6: 1～8: 1 with the ratio of the weight of the described fish-skin of hygrometric state, and the time of injecting continuously clear water was controlled in 1 hour.
(5) the continuous stripping of collagen protein: implantation temperature is 55～95 ℃ clear water continuously in the extractor, and collagen protein stripping from the hygrometric state fish-skin was flowed out to clear water and in extractor after clear water contact with fish-skin, flows out the rate of injection of controlling with clear water equal; Effluent liquid is the solution that contains collagen protein.Temperature is controlled at 55～95 ℃ in the extractor, and described clear water is 10: 1～30: 1 with the ratio of the weight of the described fish-skin of hygrometric state, and the time of injecting continuously 55～95 ℃ of clear water is controlled at 3～10 hours.
(6) concentrated: the collagen solution that step (5) is obtained uses three to follow and concentrate or ultrafiltration process concentrates, and the solid content of collagen solution is increased to more than 30%.
(7) drying: the collagen protein concentrated solution that step (6) is obtained uses lyophilize or spray-drying process to be prepared into the collagen protein powder.
The present invention only collects the albumen leach liquor that contains collagen protein owing to protein and collagen protein step-leached with non-collagen class, has therefore improved the purity of collagen protein; Because extracting temperature, control at collagen protein irreversible thermally denature critical temperature occurs, and extraction time is lower than collagen of fish skin in the sex change critical temperature generation required time of irreversible denaturation, therefore the random degradation rate that the collagen protein that extracts occurs is low or random degraded do not occur, therefore, the collagen protein that utilizes the described method of order of the present invention to prepare has purity height and molecular weight high.
Embodiment 1
(1) fish-skin is pulverized: take by weighing fish-skin 10kg, use the 5kg clear water under 30 ℃ of conditions, to soak 4 hours, filter and remove the aqueous solution, adopt pulverizer to pulverize into about 1cm * 1cm fish-skin block, be loaded into volume and be in 200 liters the extractor, the filtering net that the aperture is 1mm is equipped with in the extractor outlet.
(2) degreasing: inject continuously the isopropanol water solution of 5 volume % in the extractor, isopropanol water solution contacts with fish-skin afterwards and flows out in extractor, and the isopropanol water solution add-on is 200L, and injection length is controlled at 32 hours, and temperature is controlled at below 18 ℃.
(3) non-SUB1095 stripping: the aqueous acetic acid that injects continuously 0.01mol/L in the extractor, after contacting with fish-skin, aqueous acetic acid flows out from extractor, temperature is controlled at 15 ℃ in the extractor, and the aqueous acetic acid injection rate is 200kg, and injection length is 12 hours continuously.
(4) clean: inject continuously the clear water of clear water 60kg in the extractor, from the extractor outflow, the interior temperature of extractor was controlled at 15 ℃ after clear water contact with fish-skin, and the time of injecting continuously clear water was controlled in 1 hour.
(5) the continuous stripping of collagen protein: implantation temperature is 55 ℃ clear water continuously in the extractor, collagen protein stripping from fish-skin was flowed out to clear water and in extractor after clear water contacted with fish-skin, the clear water injection rate is 100L, and injection length is 3 hours, and temperature is controlled at 55 ℃ in the extractor.
(6) concentrated: the collagen solution that step (5) is obtained uses three to follow concentrate with ultrafiltration process and concentrate, and the solid content of collagen solution is increased to more than 20%.
(7) drying: the collagen protein concentrated solution that step (6) is obtained uses freeze-drying to make the dry state collagen protein.
Embodiment 2
(1) fish-skin is pulverized: take by weighing fish-skin 10kg, use the 5kg clear water under 30 ℃ of conditions, to soak 4 hours, filter and remove the aqueous solution, adopt pulverizer to pulverize into about 1cm * 1cm fish-skin block, be loaded into volume and be in 200 liters the extractor, the filtering net that the aperture is 1mm is equipped with in the extractor outlet.
(2) degreasing: inject continuously the isopropanol water solution of 18 volume % in the extractor, isopropanol water solution contacts with fish-skin afterwards and flows out in extractor, and the isopropanol water solution add-on is 150L, and injection length is controlled at 24 hours, and temperature is controlled at below 18 ℃.
(3) non-SUB1095 stripping: the aqueous hydrochloric acid that injects continuously 0.03mol/L in the extractor, after contacting with fish-skin, aqueous hydrochloric acid flows out from extractor, temperature is controlled at 28 ℃ in the extractor, and the aqueous hydrochloric acid injection rate is 150kg, and injection length is 8 hours continuously.
(4) clean: inject continuously the clear water of clear water 70kg in the extractor, from the extractor outflow, the interior temperature of extractor was controlled at 20 ℃ after clear water contact with fish-skin, and the time of injecting continuously clear water was controlled in 1 hour.
(5) the continuous stripping of collagen protein: implantation temperature is 80 ℃ clear water continuously in the extractor, collagen protein stripping from fish-skin was flowed out to clear water and in extractor after clear water contacted with fish-skin, the clear water injection rate is 200L, and injection length is 7 hours, and temperature is controlled at 80 ℃ in the extractor.
(6) concentrated: the collagen solution that step (5) is obtained uses three to follow concentrate with ultrafiltration process and concentrate, and the solid content of collagen solution is increased to more than 20%.
(7) drying: the collagen protein concentrated solution that step (6) is obtained uses lyophilize to make the dry state collagen protein.
Embodiment 3
(1) fish-skin is pulverized: take by weighing fish-skin 10kg, use the 5kg clear water under 30 ℃ of conditions, to soak 4 hours, filter and remove the aqueous solution, adopt pulverizer to pulverize into about 1cm * 1cm fish-skin block, be loaded into volume and be in 200 liters the extractor, the filtering net that the aperture is 1mm is equipped with in the extractor outlet.
(2) degreasing: inject continuously the n-butanol aqueous solution of 30% volume in the extractor, n-butanol aqueous solution contacts with fish-skin afterwards and flows out in extractor, and the isopropanol water solution add-on is 100L, and injection length is controlled at 18 hours, and temperature is controlled at below 18 ℃.
(3) non-SUB1095 stripping: the aqueous citric acid solution that injects continuously 0.05mol/L in the extractor, after contacting with fish-skin, aqueous citric acid solution flows out from extractor, temperature is controlled at 40 ℃ in the extractor, and the aqueous citric acid solution injection rate is 100kg, and injection length is 6 hours continuously.
(4) clean: inject continuously the clear water of clear water 80kg in the extractor, from the extractor outflow, the interior temperature of extractor was controlled at 30 ℃ after clear water contact with fish-skin, and the time of injecting continuously clear water was controlled in 1 hour.
(5) the continuous stripping of collagen protein: implantation temperature is 95 ℃ clear water continuously in the extractor, collagen protein stripping from fish-skin was flowed out to clear water and in extractor after clear water contacted with fish-skin, the clear water injection rate is 300L, and injection length is 10 hours, and temperature is controlled at 95 ℃ in the extractor.
(6) concentrated: the collagen solution that step (5) is obtained uses three to follow concentrate with ultrafiltration process and concentrate, and the solid content of collagen solution is increased to more than 20%.
(7) drying: the collagen protein concentrated solution that step (6) is obtained uses lyophilize to make the dry state collagen protein.
Detect
Detect purity and the molecular weight distribution of the prepared product collagen protein of embodiment 1-3, the method for employing is respectively amino acid composition analysis method and gel filtration method, and the result of detection is as shown in table 1:
Table 1 is executed purity, molecular weight ranges and the molecular-weight average of the prepared collagen protein of routine 1-3
| Collagen protein purity | Molecular weight distribution | Molecular-weight average | |
| Embodiment 1 | 96％ | 5000-260000 | 200000 |
| Embodiment 2 | 97％ | 5000-270000 | 220000 |
| Embodiment 3 | 98.5％ | 5000-280000 | 230000 |
Claims (6)
1. a method for preparing high purity collagen take fish-skin as raw material is characterized in that, the method may further comprise the steps successively: (1) fish-skin is pulverized; (2) degreasing; (3) non-SUB1095 stripping; (4) clean; (5) collagen protein stripping; (6) collagen solution is concentrated; (7) collagen protein is dry.
2. method according to claim 1, wherein, all carry out in extractor described step (3), (4) and (5), and described extractor has the filtration unit of holding back fish-skin.
3. method according to claim 2, wherein, described non-SUB1095 stripping step is continuous, the condition that adopts comprises: inject continuously acidic aqueous solution in described extractor, after contacting with the hygrometric state fish-skin, described acidic aqueous solution flows out from described extractor, discharge rate equates with the rate of injection of described acidic aqueous solution, temperature is controlled at 15～40 ℃ in the described extractor, the acid concentration of described acidic aqueous solution is 0.01～0.05mol/L, described acidic aqueous solution is 10: 1～20: 1 with the ratio of the weight of described hygrometric state fish-skin, and the time of injecting continuously described acidic aqueous solution is controlled in 6～12 hours scopes.
4. method according to claim 3, wherein, described acidic aqueous solution is the aqueous solution of acetic acid, citric acid or hydrochloric acid.
5. method according to claim 2, wherein, described cleaning step is continuous, the condition that adopts comprises: inject continuously clear water in described extractor, flow out from described extractor after described clear water contacts with the hygrometric state fish-skin, discharge rate equates with the rate of injection of described clear water, and the interior temperature of described extractor is controlled at 10～30 ℃, described clear water is 6: 1～8: 1 with the ratio of the weight of described hygrometric state fish-skin, and the time of injecting continuously described clear water was controlled in 1 hour.
6. method according to claim 2, wherein, described collagenic protein stripping step is continuous, the condition that adopts comprises: implantation temperature is 55～95 ℃ clear water continuously in the described extractor, described clear water is with after described hygrometric state fish-skin contacts, collagen protein from described hygrometric state fish-skin stripping to the described clear water and in described extractor, flow out, discharge rate equates with the rate of injection of described clear water, collect effluent liquid, temperature is controlled at 55～95 ℃ in the described extractor, described clear water is 10: 1～30: 1 with the ratio of the weight of the fish-skin of described hygrometric state, and the time of injecting continuously described 55～95 ℃ of clear water is controlled at 3～10 hours.
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| CN103805071A (en) * | 2014-02-14 | 2014-05-21 | 青岛华科生物技术有限公司 | Extraction method of deep-sea fish gelatin |
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| CN1749296A (en) * | 2005-10-11 | 2006-03-22 | 大连轻工业学院 | Acid soluble fish skin collagen and its preparing method |
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| CN1749296A (en) * | 2005-10-11 | 2006-03-22 | 大连轻工业学院 | Acid soluble fish skin collagen and its preparing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103805071A (en) * | 2014-02-14 | 2014-05-21 | 青岛华科生物技术有限公司 | Extraction method of deep-sea fish gelatin |
| CN103805071B (en) * | 2014-02-14 | 2015-05-20 | 青岛华科生物技术有限公司 | Extraction method of deep-sea fish gelatin |
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