CN103320486B - Method for producing chondroitin sulfate with coproduction of hydrolyzed collagen by employing fish cartilage - Google Patents
Method for producing chondroitin sulfate with coproduction of hydrolyzed collagen by employing fish cartilage Download PDFInfo
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Abstract
The invention discloses a method for producing chondroitin sulfate with coproduction of hydrolyzed collagen by employing fish cartilage. The method comprises the following steps: 1, pretreatment; 2, enzymolysis; 3, heat treatment/filtration concentration; 4, resin adsorption; 5, chondroitin sulfate refining; and 6, protein recovery. The chondroitin sulfate and collagen are simultaneously hydrolyzed by employing the composite enzyme hydrolysis technology. The collagen released from the chondroitin sulfate and the collagen in the fish cartilage are partially hydrolyzed; and moreover, lots of protein pollutants produced by the traditional chondroitin sulfate are recycled, synchronous production of the chondroitin sulfate and hydrolyzed collagen is realized, the fish cartilage is comprehensively utilized, and the method achieves energy-saving, emission-reducing purposes and has obvious economic benefits and social benefits.
Description
Technical field
The present invention relates to a kind of method that fish cartilage produces chondroitin sulfate coproduction Collagen Hydrolysate, belong to chemical technology field.
Background technology
Chondroitin sulfate (Chondroitin Sulfate is called for short CS) is the important acidic mucopolysaccharide of one being extracted preparation by the cartilaginous tissue such as bone, tracheae in animal larynx bone, nose.Chondroitin sulfate is distributed widely in people with some animals as in the tissues such as ox, sheep, pig, fish, and its main component is the mixture of the isomer such as chondroitin-4-suleate (CSA) and 6-chondroitin sulfate (CSC).Chondroitin sulfate is mainly used in treating cardiovascular and cerebrovascular disease, joint disease, and has reducing blood-fat, antithrombotic, the effect such as antitumor.China's chondroitin sulfate exports to the many countries of US and European in a large number, in the ten large class Chinese patent medicine raw materials that world market is badly in need of, occupy the 3rd.In recent years, chondroitin sulfate prevent and treat coronary heart disease, sacroiliitis and antitumor etc. in just day by day in widespread attention.
The method that current domestic chondroitin sulfate is produced mainly contains three kinds: salt solution, alkaline hydrolysis and enzymolysis process, the more method of domestic use be adopt diluted alkaline or concentrated base to combine with enzymolysis method to extract chondroitin sulfate, this alkaline process enzymolysis production process is adopted to there is complex steps, cycle longer, generally need 3-5 days, quality product and yield instability, in high temperature steaming, alkaline extraction, enzymolysis and decolorization, chondroitin sulfate is subject to havoc, product yield is on the low side, solution colour is deepened, alcohol consumption quantity is large, and production cost is high.
And a large amount of animal proteinum produced in current chondroitin sulfate production process and degraded product thereof all fail utilize and discharge, untreated organic waste water is pitch-black smelly, and eutrophication, severe contamination is brought to water surrounding.The relatively large chondroitin sulfate manufacturing enterprise of small part is only had to administer sewage at present, generally take simple physical chemistry to be treated to main end treatment mode more, afterbody process is all concentrated on by a large amount of protein pollutant, disposable input is larger, running expense is high, protein is not utilized, and waste discharge is difficult to realize up to standard.
Fish cartilage is the by product produced in China's processing of aquatic products process.Because the structure comparison of fish cartilage is loose, main component is mucopolysaccharide, protein and inorganic salt.Mucopolysaccharide alternately combines the assorted poly-polysaccharide of the acidity formed by hexosamine and hexuronic acid, and its main component is chondroitin sulfate.
Therefore consider the comprehensive utilization of fish cartilage, while production chondroitin sulfate, prepare protein powder, the added value of production can be increased, can environmental pollution be reduced again, but the current report having no this type of technology.
Summary of the invention
The present invention is in order to overcome the above-mentioned shortcoming existed in prior art, a kind of fish cartilage is provided to produce the method for chondroitin sulfate coproduction Collagen Hydrolysate, the synchronous production of chondroitin sulfate and protolysate in fish cartilage can be realized, utilize the chondroitin sulfate that production technique of the present invention is produced, purity reaches 95.6%, the protein content more than 92% of Collagen Hydrolysate, achieves the cleaning of fish cartilage, high-valued comprehensive utilization.
For achieving the above object, the technology used in the present invention solution is: a kind of fish cartilage produces the method for chondroitin sulfate coproduction Collagen Hydrolysate, and its step comprises:
One, pre-treatment: get the fresh fish-bone 3h that is soaked in water and to be placed in steam cooker 100
oc boils 30min, pulls the rear rejecting flesh of fish and grease out, rinses, be chopped into the bulk that size is 1cm × 1cm, weigh for subsequent use with clear water;
Two, enzymolysis: the aqueous solution that pretreated cartilage is added pH=8.5 by solid-liquid ratio 1:2, adds enzymic hydrolysis, constantly should remain on 8.0-8.5 by regulator solution pH in hydrolytic process;
Three, thermal treatment/filtering and concentrating: thermal treatment adds dilute hydrochloric acid or phosphoric acid adjustment solution ph in enzymolysis solution, and heating raised temperature, to solution layering, continues heating 30min, places and filter, remove non-hydrolytic acidity albumen, concentrated after solution layered filtration;
Four, resin absorption: through the continuous exchange adsorption of ion exchange resin after filtrate is concentrated, the feed liquid after exchange is protein hydrolysate and polysaccharide soln, through resin absorption after filtrate is concentrated, acetic acid-sodium acetate buffer solution wash-out, collects elutriant;
Five, POV chrondroitin: the analytical pure alcohol precipitation that elutriant adds 2 times of volumes after hydrogen peroxide for decoloration obtains chondroitin sulfate, and pelleting centrifugation is placed in thermostatic drying chamber, temperature control 60
oc is dry, and 4h obtains sterling chondroitin sulfate;
Six, reclaim albumen: the unconjugated hydrolysed protein soln after resin absorption is spray-dried, reclaim preparation protein hydrolysate powder.
Further, the judgement being hydrolyzed completeness in described step 2 is: get a little hydrolysis drop and add 50g/L Tricholroacetic Acid and observe solutions turbid degree and determine.
Further, the enzyme added in described step 2 is papoid, neutral protease and prozyme (FM), and adding proportion is 1:1:1 or 1:2:1 or 1:1:2.
Further, in described step 2, enzymolysis time is 2-6h, and hydrolysis temperature is 37-65
oc, enzyme dosage is 2-10g/kg.
Further, in described step 3, the temperature of enzymolysis postheat treatment is 70-90
oc, pH are 3.0-4.5.
Further, the resin adopted in resin absorption process in described step 6 is D001-cc resin, is loaded in 33 × 25cm chromatography columns, respectively with the acetic acid-sodium acetate buffer solution balance resin of pH6.0, pH5.0, pH4.0, identical with elutriant pH value to effluent liquid, balance completes.Loading wash-out: add above-mentioned chondroitin sulfate chromatographic solution 20mL respectively, after entering resin completely to chromatographic solution, with elutriant drip washing post jamb 2 times, then use the acetic acid-sodium acetate buffer solution wash-out of pH4.0, pH5.0, pH6.0 respectively, flow velocity 60mL/h, collect effluent liquid, added by ethanol in above-mentioned ion exchange chromatography liquid and reach 75% to ethanol content, chondroitin sulfate is separated out, filter, washing with alcohol 2 times, dries, obtains chondroitin sulfate sterling.
The invention has the beneficial effects as follows: 1, adopt combinative enzyme hydrolysis technology, realize the synchronous hydrolysis of chondroitin sulfate and collagen protein.By the collagen protein partial hydrolysis in fish cartilage while chondroitin sulfate is discharged.2, a large amount of protein pollutants produced by traditional chondroitin sulfate are recycled, and achieve the synchronous production of chondroitin sulfate and Collagen Hydrolysate, achieve the comprehensive utilization of fish cartilage, energy-saving and emission-reduction simultaneously, economic benefit and social benefit remarkable.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, to help understanding content of the present invention.
Fish cartilage produces a method for chondroitin sulfate coproduction Collagen Hydrolysate, and its step comprises:
One, pre-treatment: get the fresh fish-bone 3h that is soaked in water and to be placed in steam cooker 100
oc boils 30min, pulls the rear rejecting flesh of fish and grease out, rinses, be chopped into the bulk that size is 1cm × 1cm, weigh for subsequent use with clear water;
Two, enzymolysis: the aqueous solution that pretreated cartilage is added pH=8.5 by solid-liquid ratio 1:2, adds enzymic hydrolysis, constantly should remain on 8.0-8.5 by regulator solution pH in hydrolytic process;
Three, thermal treatment/filtering and concentrating: thermal treatment adds dilute hydrochloric acid or phosphoric acid adjustment solution ph in enzymolysis solution, and heating raised temperature, to solution layering, continues heating 30min, places and filter, remove non-hydrolytic acidity albumen, concentrated after solution layered filtration;
Four, resin absorption: through the continuous exchange adsorption of ion exchange resin after filtrate is concentrated, the feed liquid after exchange is protein hydrolysate and polysaccharide soln, through resin absorption after filtrate is concentrated, acetic acid-sodium acetate buffer solution wash-out, collects elutriant;
Five, POV chrondroitin: the analytical pure alcohol precipitation that elutriant adds 2 times of volumes after hydrogen peroxide for decoloration obtains chondroitin sulfate, and pelleting centrifugation is placed in thermostatic drying chamber, temperature control 60
oc is dry, and 4h obtains sterling chondroitin sulfate;
Six, reclaim albumen: the unconjugated hydrolysed protein soln after resin absorption is spray-dried, reclaim preparation protein hydrolysate powder.
Further, the judgement being hydrolyzed completeness in described step 2 is: get a little hydrolysis drop and add 50g/L Tricholroacetic Acid and observe solutions turbid degree and determine.
Further, the enzyme added in described step 2 is papoid, neutral protease and prozyme (FM), and adding proportion is 1:1:1 or 1:2:1 or 1:1:2.
Further, in described step 2, enzymolysis time is 2-6h, and hydrolysis temperature is 37-65
oc, enzyme dosage is 2-10g/kg.
Further, in described step 3, the temperature of enzymolysis postheat treatment is 70-90
oc, pH are 3.0-4.5.
Further, the resin adopted in resin absorption process in described step 6 is D001-cc resin, is loaded in 33 × 25cm chromatography columns, respectively with the acetic acid-sodium acetate buffer solution balance resin of pH6.0, pH5.0, pH4.0, identical with elutriant pH value to effluent liquid, balance completes.Loading wash-out: add above-mentioned chondroitin sulfate chromatographic solution 20mL respectively, after entering resin completely to chromatographic solution, with elutriant drip washing post jamb 2 times, then use the acetic acid-sodium acetate buffer solution wash-out of pH4.0, pH5.0, pH6.0 respectively, flow velocity 60mL/h, collect effluent liquid, added by ethanol in above-mentioned ion exchange chromatography liquid and reach 75% to ethanol content, chondroitin sulfate is separated out, filter, washing with alcohol 2 times, dries, obtains chondroitin sulfate sterling.
embodiment 1
Get the fresh fish-bone 3h that is soaked in water and to be placed in steam cooker 100
oCboil 30min, pull the rear rejecting flesh of fish and grease out, rinse with clear water, be chopped into the bulk that size is 1cm × 1cm, weigh for subsequent use.Select processed cartilage 50g, add the aqueous solution of pH value 8.5 by solid-liquid ratio 1:2, add papoid 5g/kg, neutral protease 5g/kg and prozyme (FM) 5g/kg respectively.45
oc enzymolysis 6h.Should continuous regulator solution pH8.0-8.5 in hydrolytic process, the desirable a little hydrolysis drop of hydrolysis completeness adds 50g/L Tricholroacetic Acid to be observed solutions turbid degree and determines.Dilute hydrochloric acid/phosphoric acid adjustment solution ph 3.0 is added, heating raised temperature to 90 in enzymolysis solution
oc solution layering, continues heating 30min, places and filter, remove non-hydrolytic acidity albumen; Concentrated after solution layered filtration.Through the continuous exchange adsorption of ion exchange resin after filtrate is concentrated, acetic acid-sodium acetate buffer solution wash-out, collects elutriant.The analytical pure alcohol precipitation that elutriant adds 2 times of volumes after hydrogen peroxide for decoloration obtains chondroitin sulfate, is deposited in centrifugally to be placed in thermostatic drying chamber, temperature control 60
oCdry 4h obtains sterling CS.Feed liquid after resin absorption is spray-dried, reclaims preparation protein hydrolysate powder.
embodiment 2
Chondroitin sulfate cation exchange chromatography technology
By ion-exchange D001-cc resin, be loaded in 33 × 25cm chromatography columns, respectively with the acetic acid-sodium acetate buffer solution balance resin of pH6.0, pH5.0, pH4.0, identical with elutriant pH value to effluent liquid, balance completes.Add chromatographic solution 20mL after chondroitin sulfate enzymolysis respectively, after entering resin completely to chromatographic solution, with elutriant drip washing post jamb 2 times, then use the acetic acid-sodium acetate buffer solution wash-out of pH4.0, pH5.0, pH6.0 respectively, flow velocity 60mL/h, collect effluent liquid, added by ethanol in above-mentioned ion-exchanging eluent and reach 75% to ethanol content, chondroitin sulfate is separated out, filter, washing with alcohol 2 times, dries, obtains chondroitin sulfate sterling.
embodiment 3
Chondroitin sulfate and Collagen Hydrolysate co-production technology
Get the fresh fish-bone 3h that is soaked in water and to be placed in steam cooker 100
oCboil 30min, pull the rear rejecting flesh of fish and grease out, rinse with clear water, be chopped into the bulk that size is 1cm × 1cm, weigh for subsequent use.Select processed cartilage 50g, add the aqueous solution of pH value 8.5 by solid-liquid ratio 1:2, add papoid 5g/kg, neutral protease 10g/kg and prozyme (FM) 5g/kg respectively.55
oc enzymolysis 5h.Should continuous regulator solution pH8.0-8.5 in hydrolytic process, the desirable a little hydrolysis drop of hydrolysis completeness adds 50g/L Tricholroacetic Acid to be observed solutions turbid degree and determines.Dilute hydrochloric acid/phosphoric acid adjustment solution ph 4.5 is added, heating raised temperature to 85 in enzymolysis solution
oc solution layering, continues heating 30min, places and filter, remove non-hydrolytic acidity albumen; Concentrated after solution layered filtration.Through the continuous exchange adsorption of ion exchange resin after filtrate is concentrated, acetic acid-sodium acetate buffer solution wash-out, collects elutriant.The analytical pure alcohol precipitation that elutriant adds 2 times of volumes after hydrogen peroxide for decoloration obtains chondroitin sulfate, is deposited in centrifugally to be placed in thermostatic drying chamber, temperature control 60
oCdry 4h obtains sterling chondroitin sulfate; Feed liquid after resin absorption is spray-dried, reclaims preparation protein hydrolysate powder.
Can be found out by above-described embodiment, the present invention has following characteristics: 1, adopt combinative enzyme hydrolysis technology, realize the synchronous hydrolysis of chondroitin sulfate and collagen protein.By the collagen protein partial hydrolysis in fish cartilage while chondroitin sulfate is discharged.2, a large amount of protein pollutants produced by traditional chondroitin sulfate are recycled, and achieve the synchronous production of chondroitin sulfate and Collagen Hydrolysate, achieve the comprehensive utilization of fish cartilage, energy-saving and emission-reduction simultaneously, economic benefit and social benefit remarkable.
The above, be only the specific embodiment of the present invention, is not limited thereto, and is anyly familiar with those skilled in the art in the technical scope that this patent discloses, and can expect change easily or replace, all should be encompassed within protection scope of the present invention.
Claims (1)
1. fish cartilage produces a method for chondroitin sulfate coproduction Collagen Hydrolysate, and it is characterized in that, step comprises:
One, pre-treatment: get the fresh fish-bone 3h that is soaked in water and to be placed in steam cooker 100 DEG C and to boil 30min, pull the rear rejecting flesh of fish and grease out, rinse with clear water, be chopped into the bulk that size is 1cm × 1cm, weigh for subsequent use;
Two, enzymolysis: the aqueous solution that pretreated cartilage is added pH=8.5 by solid-liquid ratio 1:2, adds enzymic hydrolysis, constantly should remain on 8.0-8.5 by regulator solution pH in hydrolytic process;
Three, thermal treatment/filtering and concentrating: thermal treatment adds dilute hydrochloric acid or phosphoric acid adjustment solution ph in enzymolysis solution, and heating raised temperature, to solution layering, continues heating 30min, places and filter, remove non-hydrolytic acidity albumen, concentrated after solution layered filtration;
Four, resin absorption: through the continuous exchange adsorption of ion exchange resin after filtrate is concentrated, acetic acid-sodium acetate buffer solution wash-out, collects elutriant;
Five, POV chrondroitin: the analytical pure alcohol precipitation that elutriant adds 2 times of volumes after hydrogen peroxide for decoloration obtains chondroitin sulfate, and pelleting centrifugation is placed in thermostatic drying chamber, temperature control 60 DEG C of dry 4h obtain sterling chondroitin sulfate;
Six, reclaim albumen: the unconjugated hydrolysed protein soln after resin absorption is spray-dried, reclaim preparation protein hydrolysate powder;
The judgement being hydrolyzed completeness in described step 2 is: get a little hydrolysis drop and add 50g/L Tricholroacetic Acid and observe solutions turbid degree and determine;
The enzyme added in described step 2 is papoid, neutral protease and prozyme, and adding proportion is 1: 1: 1
;
In described step 2, enzymolysis time is 2-6h, and hydrolysis temperature is 37-65 DEG C, and enzyme dosage is 2-10g/kg;
In described step 3, the temperature of enzymolysis postheat treatment is 70-90 DEG C, pH is 3.0-4.5;
The resin adopted in resin absorption process in described step 4 is D001-cc resin, be loaded in 33 × 25cm chromatography columns, use pH6.0 respectively, pH5.0, acetic acid-sodium acetate buffer balance the resin of pH4.0, identical with elutriant pH value to effluent liquid, balance completes, loading wash-out: add above-mentioned chondroitin sulfate chromatographic solution 20mL respectively, after entering resin completely to chromatographic solution, with elutriant drip washing post jamb 2 times, use pH4.0 respectively again, pH5.0, acetic acid-sodium acetate buffer the wash-out of pH6.0, flow velocity 60mL/h, collect effluent liquid, ethanol is added in above-mentioned ion exchange chromatography liquid and reach 75% to ethanol content, chondroitin sulfate is separated out, filter, washing with alcohol 2 times, dry, obtain chondroitin sulfate sterling.
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| CN201410651274.7A Division CN104450841A (en) | 2013-06-27 | 2013-06-27 | Method for producing chondroitin sulfate and co-producing hydrolyzed collagen from fish cartilage |
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| CN103497261A (en) * | 2013-10-21 | 2014-01-08 | 河北三鑫实业集团有限公司 | Chondroitin sulfate production process |
| CN103602711B (en) * | 2013-11-08 | 2016-05-18 | 青岛贝尔特生物科技有限公司 | A kind of preparation method who treats myocarditic low molecular chondroitin sulfate |
| CN103623006A (en) * | 2013-11-26 | 2014-03-12 | 青岛贝尔特生物科技有限公司 | Production method of fish cartilage extract for preventing and treating osteoarthritis |
| CN103665189A (en) * | 2013-12-10 | 2014-03-26 | 淮安麦德森制药有限公司 | Kaolin carrier method used for precipitation of chondroitin sulfate |
| CN104480168A (en) * | 2014-11-25 | 2015-04-01 | 荣成宏业海洋科技有限公司 | Method for preparing chondroitin sulfate and co-producing collagen peptide and biological calcium by using fishbone |
| CN104531817B (en) * | 2015-01-07 | 2017-12-01 | 常熟理工学院 | A kind of hyaluronic acid, chondroitin sulfate, collagen peptide, bone powder fodder and the method for soap coproduction |
| CN108752467A (en) * | 2018-06-26 | 2018-11-06 | 中国海洋大学 | A kind of method that collagen and chondroitin sulfate are extracted in fish-bone class processing fent |
| CN110144373A (en) * | 2019-05-29 | 2019-08-20 | 山东海钰生物股份有限公司 | Extract the method for chondroitin sulfate and collagen peptide respectively using animal cartilage |
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Application publication date: 20130925 Assignee: Qingdao Belt Marine Technology Co., Ltd. Assignor: Better (Qingdao) Biological Technology Co., Ltd. Contract record no.: 2014370000175 Denomination of invention: Method for producing chondroitin sulfate with coproduction of hydrolyzed collagen by employing fish cartilage License type: Common License Record date: 20141208 |
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