CN103130915A - Chondroitin sulfate preparation method based on fish head cartilage - Google Patents
Chondroitin sulfate preparation method based on fish head cartilage Download PDFInfo
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Abstract
The invention provides a chondroitin sulfate preparation method based on a fish head cartilage. The chondroitin sulfate preparation method based on the fish head cartilage comprises the following steps: taking the fish head cartilage to carry out superfine grinding; adding cartilage powder to water, carrying out ultrasonic processing, respectively adopting papain and alkaline protease to carry out enzymolysis, and obtaining enzymatic hydrolysate; adding trichloroacetic acid to the enzymatic hydrolysate, evenly mixing the enzymatic hydrolysate and the trichloroacetic acid, carrying out still standing, and collecting supernatant fluid in a centrifuging mode; adding sodium hydroxide and ethanol to the supernatant fluid, evenly mixing, stirring, and carrying out digestion; mixing to be in a neutral state, collecting sediment of the lower layer in a centrifuging mode, drying the sediment, and obtaining the fish head chondroitin sulfate. Due to the fact that the chondroitin sulfate is extracted from the fish head cartilage, fishery salvaged materials are made full use of, and added value of products is improved. A superfine grinding technology is adopted to process the fish head cartilage, and extraction efficiency of the chondroitin sulfate is improved. Meanwhile, an ultrasonic assistance compound enzymic method is adopted to process raw materials, a deproteinization effect is good, and purity of chondroitin sulfate products is high. Due to the fact that an alkali treatment precipitation method is adopted to extract the chondroitin sulfate, extraction efficiency is high.
Description
Technical field
The present invention relates to the preparation method of chondroitin sulfate, relate in particular the method for utilizing the fish head to prepare chondroitin sulfate.
Background technology
Since the 1950's, the world fisheries ultimate production increases with the speed of 6%-7% always, although fishery production surpasses 100,000,000 tons already, has nearly 1/3 catches to fail directly to be utilized by the mankind.China's fishery products total amount surpasses 5,200 ten thousand tons, becomes world fisheries and produces the first big country in continuous 20 years.Fish add trade union and produce a large amount of tankage, and the fish head is the main tankage in the fish course of processing, have occupied the 15-30% left and right of fish gross weight.A fish rich in proteins, fat, calcium, phosphorus, iron, VITAMIN, the aminoacid pattern of protein needs close to human body, is rich in unsaturated fatty acids and the phosphatide such as DHA, EPA in lipid acid, has certain value of exploiting and utilizing.
At present the exploitation of fish head mainly contained following approach: 1. directly aquatic foods freeze or freezing fish head, do the work in-process such as adult fish heads chafing dish, supply market.This method is used limited mainly for part fish head; 2. the fish head is dry, pulverizing, be ground into fishbone powder, is processed into animal-feed.Although this method is simple, the added value of product of producing is lower; 3. reclaim protein (peptide) and fish oil from the fish head, develop related products.This method Recent study is more, but related-art process is not yet ripe, does not really realize extensive commercial application.For utilizing level not high to the fish head at present, the series of problems such as processed goods is with low content of technology, added value is low is badly in need of seeking solution.
Chondroitin sulfate is a kind of of glycosaminoglycan, and as a kind of natural biological polymeric compound, chondroitin sulfate has and promotes coronary artery circulation, reducing blood-fat, antiviral, anticoagulation, the biological activity such as antitumor.In addition, chondroitin sulfate is extensive in field application prospects such as medicine, protective foods, cosmetics.At present, chondroitin sulfate is carried the cartilage from terrestrial animal such as pig and ox more.The outburst of the terrestrial animal such as mad cow disease, porcine influenza property disease makes people have a misgiving to ox source property and pig source property chondroitin sulfate series products.Also there is the people to extract chondroitin sulfate from the cartilage of marine animal shark, but because the source of raw material is limited, is difficult to meet the need of market.Also be rich in the soft element element of sulfuric acid in the fish cranial cartilage, relevant extracting method appears in the newspapers.But present extracting method complex operation needs independently to remove albumen and alcohol precipitation step, and target product content and purity are all lower, have restricted the popularization of technology and method.This patent adopts the technical matters of ultrasound-assisted enzymolysis-alkaline condition alcohol extracting, and synchronous realization is except albumen and extract chondroitin sulfate, and products therefrom purity is high, has very strong application prospect.
Summary of the invention
The present invention aims to provide a kind of method for preparing chondroitin sulfate based on the fish cranial cartilage, aims to provide a preparation method that cover efficient is high, product purity is high.
In order to achieve the above object, a kind of method for preparing chondroitin sulfate based on the fish cranial cartilage of the present invention comprises the steps:
Step1, get fish cranial cartilage micronizing, obtain fish cranial cartilage powder;
Step2, described fish cranial cartilage powder is sneaked in 3-10 water doubly with volume/weight ratio, with 300-800 watt ultrasonication 10-30 minute;
Step3, accent pH are 3-9, add papoid, 40-70 ℃ of lower enzymolysis; Transferring pH after the 90-100 ℃ of enzyme that goes out is 7-11, adds Sumizyme MP, 40-70 ℃ of lower enzymolysis, and the 90-100 ℃ of enzyme that goes out obtains enzymolysis solution;
Step4, add trichoroacetic acid(TCA) in described enzymolysis solution, final concentration is 5-20%, stirring and evenly mixing, standing 6-48 hour; Enzymolysis solution in=5000 * g under centrifugal 10-30 minute, collect supernatant liquor;
Step5, add sodium hydroxide in described supernatant liquor, final concentration is the 0.2-0.6 mol/L; 95% ethanol that adds again 2-6 times of volume ratio, mixing was in 4-8 ℃ of lower stirring and leaching 2-10 hour; After solution transfers to neutrality, in=5000 * g under centrifugal 10-30 minute, collect lower sediment; Described precipitation gets a fish chondroitin sulfate after drying.
Under optimal way, in above-mentioned Step3, papoid is selected EC3.4.22.2, and Sumizyme MP is selected EC3.4.21.14.And the add-on of papoid and Sumizyme MP is the 0.1-3.0% of fish cranial cartilage grain weight amount, and enzymolysis is 1-4 hour.The above-mentioned quantity of enzyme and the enzymolysis time of adding is of value to raising chondroitin sulfate extraction yield, better removes simultaneously albumen, improves the purity of chondroitin sulfate product.
In addition, under optimal way in step Step3 adjust pH select 1-3 mol/L citric acid or sodium hydroxide; The enzyme duration that goes out is more than 10 minutes.Optimal way all is of value to the raising product purity.
In addition, in order further boosting productivity, in step Step1, at first to get the fish head that fresh or freezing preservation, flowing water are thawed, at 70-100 ℃ of thermal treatment 10-30 minute, to cut the fish head open, take out cartilage, except pulverizing after degrease and muscle.And under optimal way, grinding particle size is (usually more than 50 microns) below 300 microns, and grinding particle size is as far as possible little here, in order to improve efficient and the product purity of subsequent operations.Should consider the cost of concrete operations simultaneously, undersized after all, running cost is corresponding raising also, and the grinding particle size that prior art can reach all is applicable to the present invention.
The present invention is based on the freshwater fish cranial cartilage and prepare the method for chondroitin sulfate, compared with prior art the present invention has following advantage:
1. extract chondroitin sulfate from the fish cranial cartilage, take full advantage of fishery wastes, the improving product added value;
2. adopt superfine communication technique treat fish cranial cartilage, improve the chondroitin sulfate extraction yield;
3. adopt ultrasonic assisted recombination enzymatic treatment raw material, effective except albumen, the chondroitin sulfate product purity is high; Adopt step alkaline purification-precipitator method to extract chondroitin sulfate, extraction efficiency is higher;
4. the utilization of this technology is conducive to turn waste into wealth, and is fit to the extensively popularization of fishery area.
Embodiment
Embodiment 1: get fresh fish head, the fish head is cut in 70 ℃ of thermal treatment 10 minutes open, takes out cartilage, except degrease and muscle, is cut into fritter below 1 centimetre, then through micronizing to granularity below 300 microns.
The water of fish cranial cartilage powder with 3 times of volumes (v/w) is mixed, with 300 watts of ultrasonication 10 minutes; Transferring pH with 1 mol/L citric acid is 3; Add papoid (EC3.4.22.2), the enzyme dosage is 0.1%, 40 ℃ of lower enzymolysis 4 hours of fish cranial cartilage grain weight amount; Be heated to 90 ℃ of enzymes 15 minutes of going out; Transferring pH with 1 mol/L sodium hydroxide is 7; Add Sumizyme MP (EC3.4.21.14), the enzyme dosage is 0.1%, 40 ℃ of lower enzymolysis 4 hours of fish cranial cartilage grain weight amount, is heated to 90 ℃ of enzymes 10 minutes of going out.
Add trichoroacetic acid(TCA) in enzymolysis solution, final concentration is 5%, stirring and evenly mixing, standing 6 hours; Enzymolysis solution under 5000 * g centrifugal 10 minutes is collected supernatant liquor; Adding sodium hydroxide in the supernatant liquor, to make its final concentration be 0.2 mol/L, then add 95% ethanol of 2 times of volumes (v/v), mixing, in 4 ℃ of lower stirring and leaching 2 hours, after solution transfers to neutrality, under 5000 * g centrifugal 10 minutes, collect lower sediment, namely get after drying a fish chondroitin sulfate.
Embodiment 2: get fresh fish head, the fish head is cut in 100 ℃ of thermal treatment 30 minutes open, takes out cartilage, except degrease and muscle, is cut into 0.5 centimetre of fritter, then through micronizing to granularity below 300 microns.
The water of fish cranial cartilage powder with 10 times of volumes (v/w) is mixed, with 800 watts of ultrasonication 30 minutes; Transferring pH with 3 mol/L sodium hydroxide is 9; Add papoid (EC3.4.22.2), the enzyme dosage is 3.0%, 70 ℃ of lower enzymolysis 1 hour of fish cranial cartilage grain weight amount; Be heated to 100 ℃ of enzymes 10 minutes of going out; Transferring pH with 1 mol/L sodium hydroxide is 11; Add EC3.4.21.14, the enzyme dosage is 3.0%, 70 ℃ of lower enzymolysis 1 hour of fish cranial cartilage grain weight amount; Be heated to 100 ℃ of enzymes 10 minutes of going out.
Add trichoroacetic acid(TCA) in enzymolysis solution, final concentration is 20%, stirring and evenly mixing, standing 6 hours; Enzymolysis solution under 10000 * g centrifugal 30 minutes is collected supernatant liquor; Adding sodium hydroxide in the supernatant liquor, to make its final concentration be 0.6 mol/L, 95% ethanol that adds again 6 times of volumes (v/v), mixing, in 8 ℃ of lower stirring and leaching 10 hours, after solution transfers to neutrality, under 10000 * g centrifugal 30 minutes, collect lower sediment, namely get after drying a fish chondroitin sulfate.
Embodiment 3: get fresh fish head, the fish head is cut in 80 ℃ of thermal treatment 20 minutes open, takes out cartilage, except degrease and muscle, is cut into 1 centimetre of fritter, then through micronizing to granularity below 300 microns;
The water of fish cranial cartilage powder with 4 times of volumes (v/w) is mixed, with 500 watts of ultrasonication 20 minutes; Transferring pH with 2 mol/L citric acids is 5; Add papoid (EC3.4.22.2), the enzyme dosage is 2.0%, 60 ℃ of lower enzymolysis 3 hours of fish cranial cartilage grain weight amount; Be heated to 100 ℃ of enzymes 10 minutes of going out; Transferring pH with 1 mol/L sodium hydroxide is 10; Add Sumizyme MP (EC3.4.21.14), the enzyme dosage is 2.0%, 50 ℃ of lower enzymolysis 2 hours of fish cranial cartilage grain weight amount; Be heated to 100 ℃ of enzymes 10 minutes of going out.Add trichoroacetic acid(TCA) in enzymolysis solution, final concentration is 10%, stirring and evenly mixing, standing 24 hours; Enzymolysis solution under 7500 * g centrifugal 20 minutes is collected supernatant liquor; Adding sodium hydroxide in the supernatant liquor, to make its final concentration be 0.3 mol/L, then add 95% ethanol of 3 times of volumes (v/v), mixing, in 6 ℃ of lower stirring and leaching 6 hours, after solution transfers to neutrality, under 8000 * g centrifugal 15 minutes, collect lower sediment, namely get after drying a fish chondroitin sulfate.
Embodiment 4: get freezing preservation, the fish head that flowing water thaws, the fish head is cut in 90 ℃ of thermal treatment 20 minutes open, takes out cartilage, except degrease and muscle, is cut into 1 centimetre of fritter, then through micronizing to granularity below 300 microns;
The water of fish cranial cartilage powder with 5 times of volumes (v/w) is mixed, with 700 watts of ultrasonication 20 minutes; Transferring pH with 3 mol/L citric acids is 5; Add papoid (EC3.4.22.2), the enzyme dosage is 0.5%, 50 ℃ of lower enzymolysis 2 hours of fish cranial cartilage grain weight amount; Be heated to 95 ℃ of enzymes 15 minutes of going out; Transferring pH with 3 mol/L sodium hydroxide is 8; Add Sumizyme MP (EC3.4.21.14), the enzyme dosage is 0.5%, 60 ℃ of lower enzymolysis 3 hours of fish cranial cartilage grain weight amount; Be heated to 95 ℃ of enzymes 15 minutes of going out.Add trichoroacetic acid(TCA) in enzymolysis solution, final concentration is 15%, stirring and evenly mixing, standing 36 hours; Enzymolysis solution under 12000 * g centrifugal 25 minutes is collected supernatant liquor; Adding sodium hydroxide in the supernatant liquor, to make its final concentration be 0.5 mol/L, 95% ethanol that adds again 4 times of volumes (v/v), mixing, in 5 ℃ of lower stirring and leaching 5 hours, after solution transfers to neutrality, under 12000 * g centrifugal 20 minutes, collect lower sediment, namely get after drying a fish chondroitin sulfate.
Embodiment 5: get freezing preservation, the fish head that flowing water thaws, the fish head is cut in 100 ℃ of thermal treatment 20 minutes open, takes out cartilage, except degrease and muscle, is cut into 0.5 centimetre of fritter, then through micronizing to granularity below 300 microns;
The water of fish cranial cartilage powder with 6 times of volumes (v/w) is mixed, with 600 watts of ultrasonication 15 minutes; Transferring pH with 1 mol/L citric acid is 3; Add papoid (EC3.4.22.2), the enzyme dosage is 1.3%, 55 ℃ of lower enzymolysis 3 hours of fish cranial cartilage grain weight amount; Be heated to 98 ℃ of enzymes 10 minutes of going out; Transferring pH with 1 mol/L sodium hydroxide is 9; Add Sumizyme MP (EC3.4.21.14), the enzyme dosage is 1.3%, 55 ℃ of lower enzymolysis 4 hours of fish cranial cartilage grain weight amount; Be heated to 98 ℃ of enzymes 10 minutes of going out.Add trichoroacetic acid(TCA) in enzymolysis solution, final concentration is 10%, stirring and evenly mixing, standing 24 hours; Enzymolysis solution under 7500 * g centrifugal 30 minutes is collected supernatant liquor; Adding sodium hydroxide in the supernatant liquor, to make its final concentration be 0.4 mol/L, then add 95% ethanol of 2 times of volumes (v/v), mixing, in 7 ℃ of lower stirring and leaching 4 hours, after solution transfers to neutrality, under 6000 * g centrifugal 10 minutes, collect lower sediment, namely get after drying a fish chondroitin sulfate.
The above; only be the better embodiment of the present invention; but protection scope of the present invention is not limited to this; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to replacement or changed according to technical scheme of the present invention and inventive concept thereof, within all should being encompassed in protection scope of the present invention.
Claims (6)
1. a method for preparing chondroitin sulfate based on the fish cranial cartilage, is characterized in that, comprises the steps:
S1, get the fish cranial cartilage and pulverize, obtain fish cranial cartilage powder;
S2, described fish cranial cartilage powder is sneaked in 3-10 water doubly with volume/weight ratio, with 300-800 watt ultrasonication 10-30 minute;
S3, accent pH are 3-9, add papoid, 40-70 ℃ of lower enzymolysis; Transferring pH after the 90-100 ℃ of enzyme that goes out is 7-11, adds Sumizyme MP, and 40-70 ℃ of lower enzymolysis obtains enzymolysis solution after the 90-100 ℃ of enzyme that goes out;
S4, add trichoroacetic acid(TCA) in described enzymolysis solution, final concentration is 5-20%, stirring and evenly mixing, standing 6-48 hour; Enzymolysis solution in=5000 * g under centrifugal 10-30 minute, collect supernatant liquor;
S5, add sodium hydroxide in described supernatant liquor, final concentration is the 0.2-0.6 mol/L; 95% ethanol that adds again 2-6 times of volume ratio, mixing was in 4-8 ℃ of lower stirring and leaching 2-10 hour; After solution transfers to neutrality, in=5000 * g under centrifugal 10-30 minute, collect lower sediment; Described precipitation gets freshwater fish head chondroitin sulfate after drying.
2. prepare according to claim 1 the method for chondroitin sulfate based on the fish cranial cartilage, it is characterized in that, papoid described in step S3 is selected EC 3.4.22.2, and described Sumizyme MP is selected EC 3.4.21.14;
The add-on of described papoid and described Sumizyme MP is the 0.1-3.0% of fish cranial cartilage grain weight amount, and enzymolysis is 1-4 hour.
3. prepare according to claim 2 the method for chondroitin sulfate based on the fish cranial cartilage, it is characterized in that, in step S3, adjust pH is selected 1-3 mol/L citric acid or sodium hydroxide.
4. prepare according to claim 3 the method for chondroitin sulfate based on the fish cranial cartilage, it is characterized in that, in step S3, the enzyme time of going out is more than 10 minutes.
5. according to claim 1-4 arbitrary described methods that prepare chondroitin sulfate based on the fish cranial cartilage, it is characterized in that, in step S1, get the fish head that fresh or freezing preservation, flowing water are thawed, at 70-100 ℃ of thermal treatment 10-30 minute, cut the fish head open, take out cartilage, except pulverizing after degrease and muscle.
6. prepare according to claim 5 the method for chondroitin sulfate based on the fish cranial cartilage, it is characterized in that, the granularity after pulverizing in step S1 is below 300 microns.
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| CN106397629A (en) * | 2016-08-30 | 2017-02-15 | 集美大学 | Method for extracting chondroitin sulfate from sturgeon bones, chondroitin sulfate extracted through method, and application of chondroitin sulfate |
| CN107114793A (en) * | 2017-04-13 | 2017-09-01 | 大连工业大学 | It is a kind of to comprehensively utilize the method that sturgeon bone prepares calcium and chondroitin sulfate |
| CN108017726A (en) * | 2018-01-31 | 2018-05-11 | 杭州赫思缇生物科技有限公司 | A kind of method from conch shell extraction chondroitin sulfate |
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| CN108913741A (en) * | 2018-05-03 | 2018-11-30 | 吉林大学 | A method of using enzymatic isolation method from pilose antler extraction purification pilose antler active oligopeptides, chondroitin sulfate |
| CN113265010A (en) * | 2021-04-15 | 2021-08-17 | 临沂欣宇辉生物科技有限公司 | Salmon proteoglycan extraction process |
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| CN103610689A (en) * | 2013-11-04 | 2014-03-05 | 鲁东大学 | Purpose of chondroitin sulfate for preparing antineoplastic immunopotentiator |
| CN103610689B (en) * | 2013-11-04 | 2016-06-08 | 鲁东大学 | Chondroitin sulfate purposes in preparing antineoplastic immune reinforcing agent |
| CN106397629A (en) * | 2016-08-30 | 2017-02-15 | 集美大学 | Method for extracting chondroitin sulfate from sturgeon bones, chondroitin sulfate extracted through method, and application of chondroitin sulfate |
| CN106397629B (en) * | 2016-08-30 | 2019-08-20 | 集美大学 | Method, the chondroitin sulfate by this method extraction and the application of chondroitin sulfate are extracted from sturgeon fish-bone |
| CN107114793A (en) * | 2017-04-13 | 2017-09-01 | 大连工业大学 | It is a kind of to comprehensively utilize the method that sturgeon bone prepares calcium and chondroitin sulfate |
| CN108017726A (en) * | 2018-01-31 | 2018-05-11 | 杭州赫思缇生物科技有限公司 | A kind of method from conch shell extraction chondroitin sulfate |
| CN108191924A (en) * | 2018-01-31 | 2018-06-22 | 杭州赫思缇生物科技有限公司 | A kind of method from prawn shell extraction chondroitin sulfate |
| CN108913741A (en) * | 2018-05-03 | 2018-11-30 | 吉林大学 | A method of using enzymatic isolation method from pilose antler extraction purification pilose antler active oligopeptides, chondroitin sulfate |
| CN113265010A (en) * | 2021-04-15 | 2021-08-17 | 临沂欣宇辉生物科技有限公司 | Salmon proteoglycan extraction process |
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