CN103773830B - A kind of method extracting collagen protein from fish scale - Google Patents
A kind of method extracting collagen protein from fish scale Download PDFInfo
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- CN103773830B CN103773830B CN201410036356.0A CN201410036356A CN103773830B CN 103773830 B CN103773830 B CN 103773830B CN 201410036356 A CN201410036356 A CN 201410036356A CN 103773830 B CN103773830 B CN 103773830B
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Abstract
A kind of method extracting collagen protein from fish scale, makes raw material with fish scale, extracts through steps such as pretreatment, dry, defat, deliming, radiation treatment, enzymolysis, extraction, purification. Owing to carrying out enzymolysis, extraction and purification with pepsin combining citric acid after fish scale raw material is carried out electron beam irradiation process, it is possible to shorten enzymolysis time, improve degree of hydrolysis, improving production efficiency, have extraction ratio height, relative molecular weight is little, purity is high, low cost of manufacture, the advantage of non-secondary pollution.
Description
Technical field
The present invention relates to a kind of collagen protein preparation method, a kind of method extracting collagen protein from fish scale specifically.
Background technology
Collagen protein (Collagen), also known as collagen, is the spirality fiber shape protein twisted into by three peptide chains. It is present in the positions such as human body skin, skeleton, tooth, tendon, and major physiological function is bonding connective tissue. Collagen has very strong biological activity and biological function, can participate in the migration of cell, differentiation and propagation, makes skeleton, tendon, cartilage and skin have certain mechanical strength. The multiformity of collagen structure and functional characteristics and complexity, determine its critical role in a lot of fields and good application prospect, and research and the application of collagen protein become a focus already. Owing to ecological environment is heavily contaminated in recent years, terrestrial mammal epidemic disease breaks out, the safety stemming from the collagen protein extracted in the animal skins such as cattle, pig, bone is under suspicion and restriction, safer fish scale collagen is all being developed energetically in countries in the world, meets people to its safety, ecological and healthy demand. Fish scale contains rich in protein and the mineral such as calcium, phosphorus, is mainly made up of protein and hydroxyapatite, and wherein protein accounts for the 50%��70% of fish scale gross weight, is mainly collagen protein and keratin, it is seen that fish scale is extraordinary collagen protein raw materials for production. Such collagen there is several amino acids, human body cell nutrition can be improved, promote the metabolism of skin and skeletal tissue, strengthen skin elasticity, make skin lubrication fine and smooth, additionally pre-preventing bone rarefaction and rheumatic arthritis, raising body immunity, enhancing vitality are had good effect. But the nutritional labeling in fish scale is forgotten by people, domestic always, substantial amounts of available fish scale is taken as garbage disposal, does not only make full use of the value of fish scale, but also contaminated environment. External that the technique extracting collagen protein from marine fishes fish scale is more ripe at present, the extraction rate reached of its collagen protein is to 30%��60%, and has applied to industrialized production. And the domestic research about extracting collagen protein in fish scale is also in initial period. Publication number is the technology that the Chinese patent of CN103333220A discloses " a kind of defat deashing method extracting collagen protein from fish scale ", and this technology, by the fish scale after processing, through a link, synchronously completes defat deliming. Simplify process procedure, it is to avoid add sodium hydroxide, reduce and pollute.But this method can reduce the extraction ratio of collagen protein. Publication number is the technology that the Chinese patent of CN101418328A discloses " preparation method of a kind of fish scale collagen ". The method is extracted by the fish scale enzymolysis solution after enzymolysis performs twice at filtration, so makes fish scale Middle nutrition composition lose few, and extraction ratio also just increases relatively. Also by salting out method precipitate and separate protein liquid, the method not only will not cause protein denaturation, and fully keeps its biological activity, so makes the purity of collagen protein relatively also increase. But the method exists the fish scale process before enzymolysis not ideal enough, and enzymolysis time is also relatively long, and degree of hydrolysis is low, and production cost is high, the defect that production efficiency is not high.
Summary of the invention
The deficiency that the present invention is contemplated to overcome in above-mentioned technology fish scale collagen yield and purity is low, it is provided that a kind of extraction ratio is high, and technique is simple, strong operability, the method extracting collagen protein from fish scale that cost is low.
The present invention seeks to be realized by following technical proposals: a kind of method extracting collagen protein from fish scale, its step is as follows:
1. adopting high-quality fish scale to make raw material, the NaOH adding 0.1mol/L soaks 24h, and distilled water is washed till neutrality, adds 0.6mol/LHCL and soaks 24h, and distilled water is washed till neutrality, removes the dope on fish scale surface and other impurity to without obvious suspended impurity;
2. dry, fish scale is dried naturally, until the water yield is less than 15%, removes impurity and uncleanly fish scale after drying, store standby;
3. defat, puts in plastic containers by above-mentioned fish scale, is soaked in water, and adds the sodium hydroxide stirring of 5%-20% fish scale weight, removes the lipid matter in fish scale, filtration and washing, adds distilled water modulation to neutral;
4. deliming, adds above-mentioned fish scale in container, soaks, and then the citric acid of the 10% of addition 5%-20% fish scale weight, stirring, till non-foam produces, slough the ash in fish scale, with distilled water flushing to neutral;
5. radiation treatment, adopts electron beam to carry out radiation treatment. Weighing and a certain amount of insert in bag through the above-mentioned fish scale processed, THICKNESS CONTROL is consistent, carries out irradiation after sealing, and irradiation dose is 5-300kGy, exposure time 0.5-5min;
6. enzymolysis, adds the water of 5-10 times of fish scale weight by the fish scale after above-mentioned electron beam irradiation, and the citric acid of 1% regulates PH to 2-3, is subsequently adding the pepsin of the 1%-5% of fish scale weight, at 5-12 DEG C continuous stirring 1.5-5 hour. Enzymolysis is warming up to more than 80 DEG C after terminating, keep carrying out enzyme denaturing in about 20 minutes;
7. extract, above-mentioned enzymolysis solution is filtered, preserve the solution after filtering, repeat filtration twice. With 4500r/min, the solution after filtering being centrifuged 20min, pours out supernatant, 3 times supernatant merges, the sodium chloride of the 5%-15% adding its weight is saltoutd, 4500r/min is centrifuged 20min, precipitates the acetic acid with the 5%-20% of its weight, repeats to saltout and dissolve operation and once obtains protein liquid;
8. purification, moves into bag filter by protein liquid, dialyses 3d with distilled water, changes water-dialyzing every day and obtain the collagen solution of purification for 2��3 times. Freeze-dried back.
The fish scale selecting high-quality makes raw material, carry out pre-treatment, thick substances and other impurity on fish scale surface is removed by acid-base method, then dry and remove raw meat, then acid-base method is adopted to carry out deliming, defat, carry out enzymolysis again through after electron beam irradiation with pepsin combining citric acid, finally carry out extracting, purification obtains high-quality fish scale collagen product.When adopting substep defat deliming, addition sodium hydroxide is possible not only to defat and could also function as the effect of loose collagen fiber compact texture, increases the extraction ratio of collagen protein. It addition, can better slough the ash in fish scale by citric acid, make fish scale more clean, also can increase collagen protein extraction ratio. So substep defat deliming should be adopted when being and obtain higher collagen protein extraction ratio as far as possible. Electron beam irradiation technology is to utilize the interaction between electron beam and material, ionizes and excite generation activated atom and anakmetomeres, and so as to material generation series of physical chemical change, cause the degraded of material, polymerization and cross-linking modified a kind of technology. Producing free radical in irradiation process, this Free Radical, in macromolecular chain, causes molecular chain rupture, produces degraded, defines the collagen protein of molecular weight, improves the absorbance of human body simultaneously, in order to people better go to utilize. Radiotechnology is typically under normal temperature and pressure conditions to carry out, and has treatment effeciency height, without adding other features such as chemical reagent and non-secondary pollution. The present invention improves the activity energy of collagen molecules by electron beam irradiation so that it is being easier to precipitate out, thus improving collagen protein extraction ratio, and can shorten enzymolysis time, improve degree of hydrolysis, reduce production cost, improves production efficiency.
Electron beam operational factor is: beam energy 6-12MeV, power 8-14kW, frequency 280-400Hz. Through experimental results demonstrate, electron beam operational factor controls can at utmost improve collagen protein activity energy in above-mentioned scope, make it easier to precipitate out, well improve collagen protein extraction ratio, and electron beam produces free radical in irradiation process, this Free Radical is in macromolecular chain, cause molecular chain rupture, produce degraded so that it is degree of hydrolysis obtains abundant raising, and then shortens enzymolysis time, improve production efficiency, reduce use cost, and the collagen that relative molecular weight is less can be obtained in its process, in order to people better utilize.
In skimming processes, the sodium hydroxide amount of being preferably added is: 8%--15%; In deliming process, the citric acid amount of being preferably added is: 6%--15%; In radiation treatment, it is preferred that irradiation dose is 100-250kGy, it is preferred that exposure time 1.5-4min.
The invention has the beneficial effects as follows: owing to the fish scale as raw material to be carried out NaOH and HCL roguing, citric acid and sodium-hydroxide method deliming defat, with carrying out enzymolysis, extraction and purification under pepsin combining citric acid after being processed by electron beam irradiation. After electron beam irradiation, it is possible to shorten enzymolysis time, improving degree of hydrolysis, improve production efficiency, have extraction ratio height, relative molecular weight is little, and purity is high, low cost of manufacture, the advantage of non-secondary pollution.
Detailed description of the invention
Below in conjunction with embodiment, the present invention will be further described.
Embodiment 1
A. adopting high-quality fish scale to make raw material, the NaOH adding 0.1mol/L soaks 24h, and distilled water is washed till neutrality, adds 0.6mol/LHCL and soaks 24h, and distilled water is washed till neutrality, removes the dope on fish scale surface and other impurity to without obvious suspended impurity;
B. dry, fish scale is dried naturally, until the water yield is less than 15%, removes impurity and uncleanly fish scale after drying, store standby;
C. defat, puts in plastic containers by above-mentioned fish scale, is soaked in water, and adds the sodium hydroxide of 10% fish scale weight, stirring, removes the lipid matter in fish scale, filtration and washing, adds distilled water modulation to neutral;
D. deliming, adds above-mentioned fish scale in container, soaks, then add 8% fish scale weight 10% citric acid, stirring, till non-foam produces, slough the ash in fish scale, with distilled water flushing to neutral;
E. radiation treatment, adopts electron beam to carry out radiation treatment. Weighing and a certain amount of insert in bag through the above-mentioned fish scale processed, THICKNESS CONTROL is consistent, carries out irradiation after sealing, and irradiation dose is 150kGy, exposure time 2min. Electron beam operational factor: beam energy is 10MeV, power 12kW, frequency 360Hz.
F. enzymolysis, adds the water of 6 times of fish scale weight by the fish scale after above-mentioned electron beam irradiation, and the citric acid of 1% regulates PH to 3, be subsequently adding fish scale weight 4% pepsin, be continuous stirring 3 hours at 10 DEG C in temperature. Enzymolysis is warming up to more than 80 DEG C after terminating, keep carrying out enzyme denaturing in about 20 minutes;
G. extract, above-mentioned enzymolysis solution is filtered, preserve the solution after filtering, repeat filtration twice. With 4500r/min, the solution after filtering being centrifuged 20min, pours out supernatant, 3 times supernatant merges, add its weight 10% sodium chloride saltout, 4500r/min is centrifuged 20min, precipitates the acetic acid with the 15% of its weight, repeats to saltout and dissolve operation and obtains protein liquid 1 time;
H. purification, moves into bag filter by protein liquid, dialyses 3d with distilled water, changes water-dialyzing every day and obtain the collagen solution of purification for 3 times. Freeze-dried back.
Embodiment 2
A. adopting high-quality fish scale to make raw material, the NaOH adding 0.1mol/L soaks 24h, and distilled water is washed till neutrality, adds 0.6mol/LHCL and soaks 24h, and distilled water is washed till neutrality, removes the dope on fish scale surface and other impurity to without obvious suspended impurity;
B. dry, fish scale is dried naturally, until the water yield is less than 15%, removes impurity and uncleanly fish scale after drying, store standby;
C. defat, puts in plastic containers by above-mentioned fish scale, is soaked in water, and adds the sodium hydroxide of 12% fish scale weight, stirring, removes the lipid matter in fish scale, filtration and washing, adds distilled water modulation to neutral;
D. deliming, adds above-mentioned fish scale in container, soaks, then add 10% fish scale weight 10% citric acid, stirring, till non-foam produces, slough the ash in fish scale, with distilled water flushing to neutral;
E. radiation treatment, adopts electron beam to carry out radiation treatment. Weighing and a certain amount of insert in bag through the above-mentioned fish scale processed, THICKNESS CONTROL is consistent, carries out irradiation after sealing, and irradiation dose is 120kGy, exposure time 2.5min. Electron beam operational factor: beam energy is 10MeV, power 12kW, frequency 360Hz.
F. enzymolysis, adds the water of 8 times of fish scale weight by the fish scale after above-mentioned electron beam irradiation, and the citric acid of 1% regulates PH to 2.5, be subsequently adding fish scale weight 3% pepsin, be continuous stirring 3 hours at 8 DEG C in temperature. Enzymolysis is warming up to more than 80 DEG C after terminating, keep carrying out enzyme denaturing in about 20 minutes;
G. extract, above-mentioned enzymolysis solution is filtered, preserve the solution after filtering, repeat filtration twice. With 4500r/min, the solution after filtering being centrifuged 20min, pours out supernatant, 3 times supernatant merges, add its weight 12% sodium chloride saltout, 4500r/min is centrifuged 20min, precipitates the acetic acid with the 18% of its weight, repeats to saltout and dissolve operation and obtains protein liquid 1 time;
H. purification, moves into bag filter by protein liquid, dialyses 3d with distilled water, changes water-dialyzing every day and obtain the collagen solution of purification for 3 times. Freeze-dried back.
Experimental data
Following table is some supplemental characteristics contrast that Electron-beam irradiation extracts collagen protein and common Enzymatic Extraction collagen protein.
As seen from the above table, the present invention utilizes the method that Electron-beam irradiation extracts collagen protein, it is possible to manufactures the collagen protein that molecular mass is little, is greatly improved collagen protein extraction ratio, reduces production cost.
Claims (2)
1. the method extracting collagen protein from fish scale, its step is as follows:
1. adopting high-quality fish scale to make raw material, the NaOH adding 0.1mol/L soaks 24h, and distilled water is washed till neutrality, adds 0.6mol/LHCL and soaks 24h, and distilled water is washed till neutrality, removes the dope on fish scale surface and other impurity to without obvious suspended impurity;
2. dry, fish scale is dried naturally, until the water yield is less than 15%, removes impurity and uncleanly fish scale after drying, store standby;
3. defat, puts in plastic containers by above-mentioned fish scale, is soaked in water, and adds the sodium hydroxide stirring of 5%-20% fish scale weight, removes the lipid matter in fish scale, filtration and washing, adds distilled water modulation to neutral;
4. deliming, adds above-mentioned fish scale in container, soaks, and then the citric acid of the 10% of addition 8%-15% fish scale weight, stirring, till non-foam produces, slough the ash in fish scale, with distilled water flushing to neutral;
5. radiation treatment, adopts electron beam to carry out radiation treatment, weighs a certain amount of through the above-mentioned fish scale processed, inserting in bag, THICKNESS CONTROL is consistent, carries out irradiation after sealing, electron beam operational factor is: beam energy 6-12MeV, power 8-14kW, frequency 280-400Hz; Irradiation dose 5-300kGy, exposure time 0.5-5min;
6. enzymolysis, fish scale after above-mentioned electron beam irradiation is added the water of 5-10 times of fish scale weight, the citric acid of 1% regulates PH to 2-3, it is subsequently adding the pepsin of the 1%-5% of fish scale weight, continuous stirring 1.5-5h at 5-12 DEG C, enzymolysis is warming up to more than 80 DEG C after terminating, keep carrying out enzyme denaturing in about 20 minutes;
7. extract, above-mentioned enzymolysis solution is filtered, preserve the solution after filtering, repeat filtration twice, the solution after filtering is centrifuged 20min with 4500r/min, pour out supernatant, 3 times supernatant merges, and the sodium chloride of the 5%-15% adding its weight is saltoutd, and 4500r/min is centrifuged 20min, precipitate the acetic acid with the 5%-20% of its weight, repeat to saltout and dissolve operation and once obtain protein liquid;
8. purification, moves into bag filter by protein liquid, dialyses 3d with distilled water, changes water-dialyzing every day and obtain the collagen solution of purification, freeze-dried back for 2��3 times.
2. a kind of method extracting collagen protein from fish scale according to claim 1, it is characterised in that in skimming processes, the sodium hydroxide amount of being preferably added is: 8%-15%; In radiation treatment, irradiation dose is 100-250kGy preferably, and exposure time is 1.5-4min preferably.
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Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103960682B (en) * | 2014-05-22 | 2015-09-23 | 上海市农业科学院 | A kind of processing method utilizing high-energy electron beam irradiation technology to improve animal protein enzymolysis efficiency |
| CN103980360A (en) * | 2014-05-29 | 2014-08-13 | 杭州市农业科学研究院 | Rapid preparation method of fish scale collagen protein |
| CN104498573A (en) * | 2014-12-11 | 2015-04-08 | 王东宇 | Method for extracting collagen from oreochromis mossambicus scales |
| CN105063154A (en) * | 2015-09-21 | 2015-11-18 | 广东海洋大学 | Method for extracting collagen from fish scales |
| CN105725109A (en) * | 2016-02-01 | 2016-07-06 | 佛山市聚成生化技术研发有限公司 | Preparation method of fish scale glue and prepared fish scale glue |
| CN106075463A (en) * | 2016-06-23 | 2016-11-09 | 袁春华 | A kind of preparation method of collagen protein medicine magnetic slow-released carrier |
| CN107022592B (en) * | 2017-03-21 | 2020-06-09 | 舟山出入境检验检疫局综合技术服务中心 | Method for extracting collagen from fish skin by low-dose irradiation |
| CN108057130A (en) * | 2017-12-26 | 2018-05-22 | 庞振方 | A kind of preparation process of medical collagen suture |
| CN108948187A (en) * | 2018-04-10 | 2018-12-07 | 华侨大学 | A kind of preparation method of fish scale enzyme dissolubility collagen |
| TWI664908B (en) * | 2018-11-22 | 2019-07-11 | 東杰國際開發有限公司 | Fish scale peeling method |
| CN109608540A (en) * | 2019-01-29 | 2019-04-12 | 广州华大生物科技有限公司 | A method of extracting collagen from fish scale |
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| CN101787078A (en) * | 2009-01-23 | 2010-07-28 | 香港百特有限公司 | Collagen polypeptide, preparation method and application thereof |
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| CN101230088A (en) * | 2008-02-22 | 2008-07-30 | 四川大学 | Method for extracting native natural collagen from animal skin or/and tendon |
| CN101307348A (en) * | 2008-06-13 | 2008-11-19 | 华中农业大学 | Method for preparing undenatured collagen from fish scale of fresh water fish |
| CN101418328A (en) * | 2008-10-28 | 2009-04-29 | 山东好当家海洋发展股份有限公司 | Method for producing fish scale collagen protein |
| CN101787078A (en) * | 2009-01-23 | 2010-07-28 | 香港百特有限公司 | Collagen polypeptide, preparation method and application thereof |
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