CN103773830A - Method for extracting collagen from fish scales - Google Patents
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Abstract
The invention relates to a method for extracting a collagen from fish scales. The method comprises the following steps: carrying out pretreatment, drying, degreasing, deliming, irradiation treatment, enzymolysis, extraction, purification and the like on the fish scales taken as raw materials, thereby extracting the collagen from the fish scales. The enzymolysis, the extraction and the purification of the fish scales taken as the raw materials are carried out by the combination of pepsins with citric acids after the fish scales are treated under the irradiation of electron beams, so that the enzymolysis time is shortened; the hydrolysis degree is improved; the production efficiency is improved. The method has the advantages of high extraction rate, small relative molecular weight, high collagen purity, low preparation cost and secondary pollution avoidance.
Description
Technical field
The present invention relates to a kind of collagen protein preparation method, is a kind of method of extracting collagen protein from fish scale specifically.
Background technology
Collagen protein (Collagen) claim again collagen, is the spirality fiber shape protein being twisted into by three peptide chains.It is present in the positions such as human body skin, bone, tooth, tendon, and major physiological function is bonding reticular tissue.Collagen has very strong biological activity and biological function, can participate in migration, the Differentiation and proliferation of cell, makes bone, tendon, cartilage and skin have certain physical strength.The diversity of collagen structure and functional characteristics and complicacy, determined its critical role in a lot of fields and good application prospect, and the research of collagen protein and application become a focus already.Because ecotope is in recent years subject to severe contamination, the outburst of Lu Sheng Mammals epidemic disease, make the security that stems from the collagen protein extracting in the animal skin such as ox, pig, bone be under suspicion and limit, safer fish scale collagen is all being developed energetically in countries in the world, meets people to its safe, ecological and healthy demand.Fish scale contains the mineral substance such as rich in protein and calcium, phosphorus, is mainly made up of protein and hydroxyapatite, and wherein protein accounts for 50%～70% of fish scale gross weight, is mainly collagen protein and Keratin sulfate, and visible fish scale is extraordinary collagen protein raw materials for production.In such collagen, there is multiple amino acids, can improve human body cell nutrition, promote the metabolism of skin and bone tissue, strengthen skin elasticity, make skin lubrication exquisiteness, in addition preventing osteoporosis and rheumatic arthritis, raising body immunity, enhancing vitality are had to good effect.But the nutritive ingredient in fish scale is forgotten by people, domestic always, a large amount of available fish scales are taken as refuse treatment, not only do not make full use of the value of fish scale, but also contaminate environment.At present abroad more ripe to extract the technique of collagen protein from marine fishes fish scale, the extraction rate reached of its collagen protein is to 30%～60%, and applied to suitability for industrialized production.And domestic about the research of extracting collagen protein in fish scale is also in initial period.Publication number is that the Chinese patent of CN103333220A discloses the technology of " a kind of degreasing deashing method that extracts collagen protein from fish scale ", and this technology, by fish scale after treatment, through a link, synchronously completes degreasing deliming.Simplify process procedure, avoided adding sodium hydroxide, reduced and pollute.But this method can reduce the extraction yield of collagen protein.Publication number is that the Chinese patent of CN101418328A discloses the technology of " a kind of preparation method of fish scale collagen ".The method is carried out twice filtration extraction by the fish scale enzymolysis solution to after enzymolysis, makes like this nutritive ingredient in fish scale lose less, and extraction yield also just increases relatively.Also have by salting-out process precipitate and separate protein liquid, the method not only can not cause protein denaturation, and fully keeps its biological activity, makes like this purity of collagen protein relatively increase yet.It is not ideal enough that but the method exists the fish scale processing before enzymolysis, 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 present invention is exactly that technique is simple, strong operability, the method for extracting collagen protein from fish scale that cost is low in order to overcome fish scale collagen yield and the low deficiency of purity in above-mentioned technology, to provide a kind of extraction yield high.
The present invention seeks to be realized by following technical proposals: a kind of method of extracting collagen protein from fish scale, its step is as follows:
1. adopt high-quality fish scale to make raw material, add the NaOH of 0.1mol/L to soak 24h, distilled water is washed till neutrality, then adds 0.6mol/L HCL to soak 24h, and distilled water is washed till neutrality, removes dope and the extremely obviously suspended impurity of nothing of other impurity on fish scale surface;
2. dry, fish scale is dried naturally, until the water yield is less than 15%, after drying, remove impurity and uncleanly fish scale, store for subsequent use;
3. degreasing, puts into plastic containers by above-mentioned fish scale, is soaked in water, and adds the sodium hydroxide of 5%-20% fish scale weight to stir, and removes the lipid matter in fish scale, filtration and washing, and adding distil water is modulated to neutrality;
4. deliming, adds above-mentioned fish scale in container, soaks, and then adds 10% citric acid of 5%-20% fish scale weight, stirs, until non-foam produces, sloughs the ash content in fish scale, with distilled water flushing to neutral;
5. radiation treatment, adopts electron beam to carry out radiation treatment.Take the fish scale of the above-mentioned processing of a certain amount of process, insert in bag, gauge control is consistent, carries out irradiation after sealing, and irradiation dose is 5-300kGy, irradiation time 0.5-5min;
6. enzymolysis, adds 5-10 doubly to the water of fish scale weight on the fish scale after above-mentioned electron beam irradiation, and 1% citric acid regulates PH to 2-3, then adds the stomach en-of the 1%-5% of fish scale weight, continuously stirring 1.5-5 hour at 5-12 ℃.After enzymolysis finishes, be warming up to more than 80 ℃, keep about the 20 minutes enzymes that go out;
7. extract, above-mentioned enzymolysis solution is filtered, preserve the solution after filtering, repeat to filter twice.By filter after solution with the centrifugal 20mi n of 4500r/min, incline and supernatant liquor, 3 times supernatant liquor merges, add the sodium-chlor of the 5%-15% of its weight to saltout, the centrifugal 20min of 4500r/min, precipitation is dissolved with the acetic acid of the 5%-20% of its weight, and repeating to saltout and dissolving operation once obtains protein liquid;
8. purifying, moves into dialysis tubing by protein liquid, uses distill water dialysis 3d, changes water-dialyzing every day and obtain for 2～3 times the collagen solution of purifying.Freeze-dried back.
Select the fish scale of high-quality to make raw material, carry out pre-treatment, remove thick substances and other impurity on fish scale surface by acid-base method, then the dry raw meat that goes, then adopt acid-base method to carry out deliming, degreasing, again by carrying out enzymolysis with stomach en-combining citric acid after electron beam irradiation, finally extract, purifying obtains high-quality fish scale collagen product.In the time adopting substep degreasing deliming, add sodium hydroxide not only can degreasing can also play loose collegen filament dense structure, increase the extraction yield of collagen protein.In addition, can better slough the ash content in fish scale by citric acid, make fish scale more clean, also can increase collagen protein extraction yield.Think and while obtaining higher collagen protein extraction yield, should adopt as far as possible substep degreasing deliming.Electron beam irradiation technology is the interaction utilizing between electron beam and material, ionizes and excites to produce activated atom and activated molecule, and make it and material generation series of physical chemical transformation, causes the degraded, polymerization of material and cross-linking modified a kind of technology.In irradiation process, produce free radical, this Free Radical, in macromolecular chain, causes molecular rupture simultaneously, produces degraded, has formed the collagen protein of molecular weight, improves the specific absorption of human body, so that people better go to utilize.Radiotechnology is generally carried out under normal temperature and pressure conditions, have processing efficiency high, without adding the features such as other chemical reagent and non-secondary pollution.The present invention improves the activity energy of collagen molecules by electron beam irradiation, it is more easily separated out, thereby improves collagen protein extraction yield, and can shorten enzymolysis time, improves degree of hydrolysis, reduces production costs, and enhances productivity.
Electron beam operating parameter is: beam energy 6-12MeV, power 8-14kW, frequency 280-400Hz.Through experimental results demonstrate, electron beam operating parameter is controlled in above-mentioned scope can at utmost improve the active energy of collagen protein, it is more easily separated out, well improve collagen protein extraction yield, and electron beam produces free radical in irradiation process, this Free Radical is in macromolecular chain, cause molecular rupture, produce degraded, make its degree of hydrolysis obtain abundant raising, and then shortened enzymolysis time, improve production efficiency, reduce use cost, and can obtain the collagen that relative molecular weight is less in its process, so that people's better utilised.
In skimming processes, the preferred add-on of sodium hydroxide is: 8%--15%; In deliming process, the preferred add-on of citric acid is: 6%--15%; In radiation treatment, preferred irradiation dose is 100-250kGy, preferred irradiation time 1.5-4min.
The invention has the beneficial effects as follows: because the fish scale to as raw material carries out NaOH and HCL impurity elimination, citric acid and the degreasing of sodium hydroxide method deliming, process rear with carrying out enzymolysis, extraction and purifying under stomach en-combining citric acid by electron beam irradiation.After electron beam irradiation, can shorten enzymolysis time, improve degree of hydrolysis, enhance productivity, there is extraction yield high, relative molecular weight is little, and purity is high, low cost of manufacture, the advantage of non-secondary pollution.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.
Embodiment 1
A. adopt high-quality fish scale to make raw material, add the NaOH of 0.1mol/L to soak 24h, distilled water is washed till neutrality, then adds 0.6mol/L HCL to soak 24h, and distilled water is washed till neutrality, removes dope and the extremely obviously suspended impurity of nothing of other impurity on fish scale surface;
B. dry, fish scale is dried naturally, until the water yield is less than 15%, after drying, remove impurity and uncleanly fish scale, store for subsequent use;
C. degreasing, puts into plastic containers by above-mentioned fish scale, is soaked in water, and adds the sodium hydroxide of 10% fish scale weight, stirs, and removes the lipid matter in fish scale, filtration and washing, and adding distil water is modulated to neutrality;
D. deliming, adds above-mentioned fish scale in container, soaks, and then adds 10% citric acid of 8% fish scale weight, stirs, until non-foam produces, sloughs the ash content in fish scale, with distilled water flushing to neutral;
E. radiation treatment, adopts electron beam to carry out radiation treatment.Take the fish scale of the above-mentioned processing of a certain amount of process, insert in bag, gauge control is consistent, carries out irradiation after sealing, and irradiation dose is 150kGy, irradiation time 2min.Electron beam operating parameter: beam energy is 10MeV, power 12kW, frequency 360Hz.
F. enzymolysis, adds 6 times to the water of fish scale weight by the fish scale after above-mentioned electron beam irradiation, and 1% citric acid regulates PH to 3, then adds 4% stomach en-of fish scale weight, is continuously stirring 3 hours at 10 ℃ in temperature.After enzymolysis finishes, be warming up to more than 80 ℃, keep about the 20 minutes enzymes that go out;
G. extract, above-mentioned enzymolysis solution is filtered, preserve the solution after filtering, repeat to filter twice.Solution after filtering, with the centrifugal 20min of 4500r/min, is inclined and supernatant liquor, and 3 times supernatant liquor merges, add 10% sodium-chlor of its weight to saltout, the centrifugal 20min of 4500r/min, precipitation is dissolved with 15% acetic acid of its weight, repeats to saltout and dissolve to operate to obtain protein liquid 1 time;
H. purifying, moves into dialysis tubing by protein liquid, uses distill water dialysis 3d, changes water-dialyzing every day and obtain for 3 times the collagen solution of purifying.Freeze-dried back.
Embodiment 2
A. adopt high-quality fish scale to make raw material, add the NaOH of 0.1mol/L to soak 24h, distilled water is washed till neutrality, then adds 0.6mol/L HCL to soak 24h, and distilled water is washed till neutrality, removes dope and the extremely obviously suspended impurity of nothing of other impurity on fish scale surface;
B. dry, fish scale is dried naturally, until the water yield is less than 15%, after drying, remove impurity and uncleanly fish scale, store for subsequent use;
C. degreasing, puts into plastic containers by above-mentioned fish scale, is soaked in water, and adds the sodium hydroxide of 12% fish scale weight, stirs, and removes the lipid matter in fish scale, filtration and washing, and adding distil water is modulated to neutrality;
D. deliming, adds above-mentioned fish scale in container, soaks, and then adds 10% citric acid of 10% fish scale weight, stirs, until non-foam produces, sloughs the ash content in fish scale, with distilled water flushing to neutral;
E. radiation treatment, adopts electron beam to carry out radiation treatment.Take the fish scale of the above-mentioned processing of a certain amount of process, insert in bag, gauge control is consistent, carries out irradiation after sealing, and irradiation dose is 120kGy, irradiation time 2.5min.Electron beam operating parameter: beam energy is 10MeV, power 12kW, frequency 360Hz.
F. enzymolysis, adds 8 times to the water of fish scale weight by the fish scale after above-mentioned electron beam irradiation, and 1% citric acid regulates PH to 2.5, then adds 3% stomach en-of fish scale weight, is continuously stirring 3 hours at 8 ℃ in temperature.After enzymolysis finishes, be warming up to more than 80 ℃, keep about the 20 minutes enzymes that go out;
G. extract, above-mentioned enzymolysis solution is filtered, preserve the solution after filtering, repeat to filter twice.Solution after filtering, with the centrifugal 20min of 4500r/min, is inclined and supernatant liquor, and 3 times supernatant liquor merges, add 12% sodium-chlor of its weight to saltout, the centrifugal 20min of 4500r/min, precipitation is dissolved with 18% acetic acid of its weight, repeats to saltout and dissolve to operate to obtain protein liquid 1 time;
H. purifying, moves into dialysis tubing by protein liquid, uses distill water dialysis 3d, changes water-dialyzing every day and obtain for 3 times the collagen solution of purifying.Freeze-dried back.
Experimental data
Following table is some supplemental characteristic contrasts that electron beam irradiation method is extracted collagen protein and common Enzymatic Extraction collagen protein.
As seen from the above table, the present invention utilizes electron beam irradiation method to extract the method for collagen protein, can manufacture the collagen protein that molecular mass is little, greatly improves collagen protein extraction yield, has reduced production cost.
Claims (3)
1. a method of extracting collagen protein from fish scale, its step is as follows:
1. adopt high-quality fish scale to make raw material, add the NaOH of 0.1mol/L to soak 24h, distilled water is washed till neutrality, then adds 0.6mol/L HCL to soak 24h, and distilled water is washed till neutrality, removes dope and the extremely obviously suspended impurity of nothing of other impurity on fish scale surface;
2. dry, fish scale is dried naturally, until the water yield is less than 15%, after drying, remove impurity and uncleanly fish scale, store for subsequent use;
3. degreasing, puts into plastic containers by above-mentioned fish scale, is soaked in water, and adds the sodium hydroxide of 5%-20% fish scale weight to stir, and removes the lipid matter in fish scale, filtration and washing, and adding distil water is modulated to neutrality;
4. deliming, adds above-mentioned fish scale in container, soaks, and then adds 10% citric acid of 5%-20% fish scale weight, stirs, until non-foam produces, sloughs the ash content in fish scale, with distilled water flushing to neutral;
5. radiation treatment, adopts electron beam to carry out radiation treatment, takes the fish scale of the above-mentioned processing of a certain amount of process, inserts in bag, and gauge control is consistent, carries out irradiation after sealing, and irradiation dose is 5-300kGy, irradiation time 0.5-5min;
6. enzymolysis, add 5-10 doubly to the water of fish scale weight on the fish scale after above-mentioned electron beam irradiation, 1% citric acid regulates PH to 2-3, then add the stomach en-of the 1%-5% of fish scale weight, continuously stirring 1.5-5 hour at 5-12 ℃, after enzymolysis finishes, be warming up to more than 80 ℃, keep about the 20 minutes enzymes that go out;
7. extract, above-mentioned enzymolysis solution is filtered, preserve filter after solution, repeat to filter twice, by filter after solution with the centrifugal 20min of 4500r/min, incline and supernatant liquor, 3 times supernatant liquor merges, and adds the sodium-chlor of the 5%-15% of its weight to saltout, the centrifugal 20min of 4500r/min, precipitation is dissolved with the acetic acid of the 5%-20% of its weight, and repeating to saltout and dissolving operation once obtains protein liquid;
8. purifying, moves into dialysis tubing by protein liquid, uses distill water dialysis 3d, changes water-dialyzing every day and obtain for 2～3 times the collagen solution of purifying, freeze-dried back.
2. a kind of method of extracting collagen protein from fish scale according to claim 1, is characterized in that electron beam operating parameter is: beam energy 6-12MeV, power 8-14kW, frequency 280-400Hz.
3. a kind of method of extracting collagen protein from fish scale according to claim 1, is characterized in that in skimming processes, the preferred add-on of sodium hydroxide is: 8%--15%; In deliming process, the preferred add-on of citric acid is: 6%--15%; In radiation treatment, irradiation dose is 100-250kGy, irradiation time 1.5-4min.
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Cited By (11)
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| CN103960682A (en) * | 2014-05-22 | 2014-08-06 | 上海市农业科学院 | Processing method for improving enzymolysis efficiency of animal protein by utilizing high energy electron beam irradiation technology |
| 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 |
| CN107022592A (en) * | 2017-03-21 | 2017-08-08 | 舟山出入境检验检疫局综合技术服务中心 | A kind of method that low dose irradiation extracts collagen in fish-skin |
| 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 |
| 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 |
| TWI664908B (en) * | 2018-11-22 | 2019-07-11 | 東杰國際開發有限公司 | Fish scale peeling method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103960682A (en) * | 2014-05-22 | 2014-08-06 | 上海市农业科学院 | Processing method for improving enzymolysis efficiency of animal protein by utilizing high energy electron beam irradiation technology |
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| 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 |
| CN107022592A (en) * | 2017-03-21 | 2017-08-08 | 舟山出入境检验检疫局综合技术服务中心 | A kind of method that low dose irradiation extracts collagen in fish-skin |
| 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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