CN102533915B - Method for preparing chondroitin sulfate and collagen polypeptide from animal cartilages - Google Patents
Method for preparing chondroitin sulfate and collagen polypeptide from animal cartilages Download PDFInfo
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Abstract
The invention discloses a method for preparing chondroitin sulfate and collagen polypeptide from animal cartilages, which comprises the following steps: crushing animal cartilage materials to obtain cartilage powder; hydrolyzing the cartilage powder with protease, and then carrying out ultrafiltration and concentration to obtain a concentrated solution; passing the concentrated solution through an anion exchange resin column, and collecting an effluent solution A; eluting with pure water, and collecting a water washing solution B; merging the effluent solution A and the water washing solution B, carrying out ultrafiltration and concentration, and carrying out spray drying to obtain the collagen polypeptide; and eluting the anion exchange resin column with an NaCl solution, carrying out ultrafiltration and concentration on the eluent solution, carrying out alcohol precipitation, dehydrolyzing, and drying to obtain the chondroitin sulfate. The invention realizes the high-valued utilization of cartilage resources, can simultaneously and efficiently extract and separate the chondroitin sulfate and the collagen polypeptide, increases the added values of the products and reduces the environment pollution.
Description
(1) technical field
The present invention relates to a kind of processing technology of animal cartilage, be specifically related to a kind of enzymolysis process and extract and adopt the directly technique of separation and purification chondroitin sulfate and collagen polypeptide from the cartilage enzymolysis solution of anionite-exchange resin.
(2) background technology
China is large agricultural country, the processing of all kinds of meat products produce tankage---the cartilage resource is very abundant, and kind is more.Be rich in chondroitin sulfate and collagen protein isoreactivity composition in animal cartilage, such bioactive macromolecule is widely used in the fields such as medicine, foods and cosmetics.Chondroitin sulfate (Chondroitin Sulfate, CS) acidic mucopolysaccharide that the repetition disaccharide unit that consists of D-Glucose aldehydic acid and N-acetylamino galactosamine forms, be mainly used in clinically the diseases such as anti-curing arthritis, treatment neurodynia, arthrodynia, arteriosclerosis, dysacousis and hepatitis, coronary heart disease, rheumatic inflammation and wound healing etc. are also had good curative effect.Collagen protein is that the animal body intensive amount is maximum, the widest protein that distributes, and extensively is present in skin, bone, tooth, tendon ligament and the blood vessel of animal.Research both at home and abroad shows, the collagen polypeptide of collagen protein and hydrolysis gained promote healthy, improve immunizing power and the aspect such as delay senility has vital role.
Domestic production enterprise mostly extracts take cartilage as raw material and isolating chondroitin sulfate, accounts for the collagen protein of total protein 90% in cartilage by enzymolysis and sex change, can only as protein fodder or with discharge of wastewater, added value of product is lower; And the raw material that extracts collagen protein and collagen polypeptide fails to realize the higher value application of cartilage resource still take os osseum, animal skin and fish scale as main.In recent years, the comprehensive utilizating research of cartilage resource has obtained extensive attention.Patent CN 1896262, CN 101913583, by enzymolysis, alcohol precipitation, reclaim respectively pure hypostasis and obtain chondroitin sulfate, reclaim the bone slag and obtain high-calcium powder, the dry collagen polypeptide that obtains of supernatant concentration.Patent CN 101063161, by enzymolysis, alcohol precipitation, obtains chondroitin sulfate, the bone slag after secondary enzymolysis, dry collagen polypeptide.Patent CN 101041842, by add the conjugated protein precipitating inhibitor in enzymolysis solution, obtains chondroitin sulfate and collagen polypeptide successively.In addition, patent CN 1721546A, CN101492491A, CN 101986859A etc. carries out concentrate drying with enzymolysis solution, obtains the mixture of chondroitin sulfate and collagen polypeptide.Above-mentioned technique has all realized the comprehensive utilization of cartilage to a certain extent, but the part that also comes with some shortcomings: (1) mixture complicated component, product applications is limited to; (2) utilize the bone slag after the cartilage enzymolysis to extract collagen polypeptide, technique is loaded down with trivial details, and in the bone slag, collagen polypeptide content is few; (3) by the enzymolysis solution alcohol precipitation is obtained chondroitin sulfate, foreign protein content is higher.
Ion exchange method is according to acid-base properties of compound in moving phase, polarity with to the difference of ion-exchanger avidity, by changing ionic strength and pH value, the separate substance successively of elutriant.Under neutrality or acidic conditions, resin anion(R.A) adsorbs in conjunction with the chondroitin sulfate polyanion, and protein polypeptide is not adsorbed.Ion exchange resin has the advantages such as selectivity is good, equipment is simple, easy to operate.The anionresin technology is applied to separation and purification chondroitin sulfate and collagen polypeptide from the cartilage enzymolysis solution, can realizes the higher value application of cartilage, the method has no report.This method has solved that the prepared using that exists in the traditional technology is insufficient, product is single or the problem such as collagen content is lower, and can significantly reduce the ethanol consumption, has broad application prospects.
(3) summary of the invention
The cartilage that the present invention produces take the processing of all kinds of meat products, as raw material, adopts enzymolysis-anionresin technology, extracts simultaneously isolating chondroitin sulfate and collagen polypeptide bioactive macromolecule from animal cartilage, has realized the higher value application of cartilage.Simultaneously, simplify production technique, reduce production costs, environmental contamination reduction.
The technical solution used in the present invention is as follows:
A kind of method of utilizing animal cartilage to prepare chondroitin sulfate and collagen polypeptide, described method is: the animal cartilage raw material pulverizing is obtained cartilage powder, cartilage powder with protease hydrolysis after ultrafiltration and concentration obtain concentrated solution, concentrated solution is crossed anionite-exchange resin, collect effluent liquid A, then use the pure water wash-out, collect water lotion B, ultrafiltration and concentration after effluent liquid A and water lotion B merge, then spraying drying makes collagen polypeptide; Anion-exchange resin column is used the NaCl eluant solution, the elutriant ultrafiltration and concentration makes chondroitin sulfate by alcohol precipitation, dehydration, drying again.
Further, said method comprising the steps of:
(1) be 0.1-10mm with animal cartilage raw material pulverizing to median size, obtain cartilage powder; Cartilage powder adds proteolytic enzyme and water, regulates the extremely optimum pH of described proteolytic enzyme of pH value, carries out enzyme digestion reaction, the controlled enzymatic hydrolysis temperature between 30-55 ℃, stirring reaction 2-12h; Then heat the enzyme that goes out, cooled and filtered is got filtrate, and adjust pH is neutral, and adopting molecular weight cut-off is that the daltonian ultra-filtration membrane ultrafiltration and concentration of 1000-8000 obtains concentrated solution, and in described concentrated solution, the mass percentage concentration of chondroitin sulfate is 1～20%; Described proteolytic enzyme is one of following: neutral protease, Sumizyme MP, papoid, bromeline or aspartic protease; The consumption of described proteolytic enzyme is the 0.1-1.5% of cartilage powder quality; The consumption of water is cartilage powder quality 5-30 times;
(2) step (1) gained concentrated solution adjust pH, to 2.0-7.0, is crossed anion-exchange resin column, collects effluent liquid A; Use the pure water wash-out, collect water lotion B, effluent liquid A and water lotion B merge, and regulating pH value is neutrality, the employing molecular weight cut-off be the daltonian ultra-filtration membrane ultrafiltration and concentration of 1000-8000 to the mass concentration of albumen be 5-30%, then spraying drying obtains collagen polypeptide;
(3) anion-exchange resin column of step (2) is eluent with 1-6mol/L NaCl solution again, chondroitin sulfate is carried out wash-out, collect elutriant, it is neutral regulating the pH value, to adopt molecular weight cut-off be the daltonian ultra-filtration membrane ultrafiltration and concentration of 1000-8000 to the mass concentration of chondroitin sulfate be 1-30%, through alcohol precipitation, dehydration, drying, make chondroitin sulfate.
Described animal cartilage raw material is that water content is lower than the animal cartilage of 15wt% after drying or drying, and described animal cartilage can be shark suft bone, hog snout bone, pig larynx bone, nose of an ox bone or pigeon breast bone etc.
In described step (1), described adjusting pH value, to the optimum pH of described proteolytic enzyme, refers to select the highest pH scope of its reactive behavior according to the concrete proteolytic enzyme that uses in reaction.The present invention's proteolytic enzyme used is all known commercial enzyme, and its optimum response pH value as well known to those skilled in the art, should make suitable selection according to the real reaction situation.More specifically, the optimum pH of neutral protease is 6.0～8.0, and the optimum pH of Sumizyme MP is 8.5～10.5, and the optimum pH of papoid is 6.0～7.0, the optimum pH of bromeline is 6.0～6.8, and the optimum pH of aspartic protease is 2.5～6.0.
Further, the vigor of the proteolytic enzyme that uses in the embodiment of the present invention is respectively: Sumizyme MP is 200,000 U/g, and bromeline is 2400 GDU/g, and aspartic protease is 50,000 U/g, and neutral protease is 200,000 U/g, and papoid is 650,000 U/g.
In described step (1), described heating is gone out enzyme normally at 70-100 ℃, insulation 10-60min, and enzyme goes out.
Described proteolytic enzyme is preferably aspartic protease, papoid or neutral protease, most preferably neutral protease.
The reagent that adjusting pH value of the present invention adopts is generally NaOH solution or HCl solution, and those skilled in the art can select suitable reagent to regulate according to actual pH and required pH value.
In described step (2), step (1) gained concentrated solution adjust pH is to 2.0-7.0, and preferred adjust pH is 3.0-5.0.
Described anionite-exchange resin is one of following: D315, D202, D307, D208, D215, D218, D301 or D354; Be preferably one of following: D218, D202, D307, D215 or D354.
The wet resin volume of described anionite-exchange resin is counted 10-30L/kg with the quality of chondroitin sulfate in concentrated solution, is preferably 15-20L/kg.
In described step (1) in concentrated solution the mass percentage concentration of chondroitin sulfate be 1～20% and step (3) in ultrafiltration and concentration to the mass concentration of chondroitin sulfate be 1-30%, here the mass percentage concentration of chondroitin sulfate can adopt potentiometric titration to follow the tracks of detection in real time, is concentrated into desired concn and gets final product.
In described step (2), described concentrated solution adjust pH, to 2.0-7.0, is crossed anion-exchange resin column, and the described flow velocity of crossing anionite-exchange resin is 0.1-10VB/h, is preferably 1-5VB/h.
In described step (2), use the pure water wash-out, described pure water is 0.1-15VB/h by the flow velocity of anionite-exchange resin, is preferably 1-10VB/h.
Described pure water typically refers to deionized water, distilled water or medicinal water, is that those skilled in the art commonly use.
The consumption of described pure water is 0.1-10 times of column volume, is preferably 2-5 times of column volume.
In described step (3), eluent is 1-6mol/L NaCl solution, preferably uses 2-3mol/L NaCl solution.The consumption of NaCl solution is 1-20 times of column volume, is preferably 5-15 times of column volume.NaCl solution is 0.1-15VB/h by the flow velocity of anionite-exchange resin, is preferably 1-10VB/h.
In described step (3), ultrafiltration and concentration is 1-30% to the mass concentration of chondroitin sulfate, and the mass concentration that preferably is concentrated into chondroitin sulfate is 2-10%.
In described step (3), described alcohol precipitation, dehydration, drying can operate usually by the following method: adding while stirring volume is concentration expressed in percentage by volume 95% aqueous ethanolic solution of 3～4 times of the concentrated solution volume after ultrafiltration and concentration, get precipitation, add again concentration expressed in percentage by volume 95% aqueous ethanolic solution dehydration, finally, vacuum-drying obtains chondroitin sulfate.This is to well known to a person skilled in the art alcohol precipitation, dehydration technique.The volumetric usage of 95% aqueous ethanolic solution of described dehydration use is counted 6～10L/kg with the chondroitin sulfate quality usually.
It is the daltonian ultra-filtration membrane of 1000-8000 that ultrafiltration and concentration of the present invention can adopt molecular weight cut-off, is preferably the daltonian ultra-filtration membrane of 3000-6000.
The present invention adopts enzymolysis and extraction, anionresin technology and ultra-filtration technique co-producing high-purity chondroitin sulfate and collagen polypeptide, and compared with prior art, its beneficial effect is embodied in:
(1) realize the higher value application of cartilage resource, high efficiency extraction isolating chondroitin sulfate and collagen polypeptide, improve added value of product simultaneously.
(2) improve the quality of chondroitin sulfate product.Anionite-exchange resin have adsorption selectivity to chondroitin sulfate, can significantly reduce the content of foreign protein in the chondroitin sulfate product.
(3) improve the quality of collagen polypeptide product.The collagen polypeptide color and luster of this technique coproduction is white, free from extraneous odour, ash content are low, molecular weight ranges is than homogeneous, concentrates and is distributed in below 4000 dalton.Collagen polypeptide stability and the biological activity of this molecular weight ranges are good, easily are absorbed, and can effectively supplement the oxyproline that human body can't independently synthesize.
(4) reclaim by albumen, environmental contamination reduction, economic benefit and environmental benefit are obvious.
(5) reduced energy consumption and production cost, operational path is short, and is simple to operate.
To sum up, the inventive method has realized the comprehensive utilization of cartilage resource, have the advantages such as extraction yield is high, adsorption selectivity good, product purity is high, the ethanol consumption is lower, technique simple, pollution is little, be conducive to economize on resources and reduce energy consumption, promote the sustainable development of animal extracts industry.
(4) description of drawings
Fig. 1 is the high-efficient liquid phase chromatogram of the collagen polypeptide sample that obtains according to the embodiment of the present invention 1.
Fig. 2 is the high-efficient liquid phase chromatogram of molecular weight standard product Exenatide (molecular weight is 4186.6, purity 〉=98%, the biochemical (Shanghai) Co., Ltd. of gill).
(5) embodiment
Below with specific embodiment explanation technical scheme of the present invention, but protection scope of the present invention is not limited to this.
The testing conditions of chondroitin sulfate described in following examples and collagen polypeptide and method of calculation are:
The qualitative and quantitative analysis of chondroitin sulfate product:, take commercially available chondroitin sulfate standard substance as contrast, carry out efficient liquid phase chromatographic analysis, appearance time and standard substance are basically identical.Quality/the quality product (dry product) * 100 of chondroitin sulfate in the content (%) of chondroitin sulfate=product in described chondroitin sulfate product; The quality of chondroitin sulfate/cartilage raw materials quality (1-water content) * 100 in the yield of chondroitin sulfate (%)=product in described product; The typical curve (relation between peak area and concentration) that in product, the quality of chondroitin sulfate is drawn with the chondroitin sulfate standard substance calculates.Foreign protein content in described chondroitin sulfate product adopts the Lowry method to detect, take commercially available bovine serum albumin standard substance as contrast.
The qualitative and quantitative analysis of collagen polypeptide product: oxyproline is the feature amino acid of collagen protein,, take commercially available oxyproline standard substance as contrast, adopts the content of Woessner colorimetric determination oxyproline.Nitrogen content adopts Kjeldahl determination to detect.The content (%) of total protein=product nitrogen content * 6.25/ quality product (dry product) * 100 in described collagen polypeptide product; The yield of total protein (%)=product nitrogen content in described product * 6.25/ cartilage raw materials quality (1-water content) * 100.The molecular weight distribution of product adopts the high performance size exclusion chromatography method to measure, testing conditions is as follows: pillar is TSK-GEL G2000SWXL chromatographic column (300mm * 7.8mm, 5 μ m), room temperature, moving phase is 45% acetonitrile (containing 0.1% trifluoroacetic acid solution), flow velocity 0.8mLmin
-1, detect wavelength 214nm.In described collagen polypeptide product, the analytical procedure of ash oontent is with reference to GB/T 5009.4-2003 (mensuration of ash content in food).
In cartilage raw material and collagen polypeptide, the analysis of aminoacid component adopts automatic analyzer for amino acids (HITACHI, L-8900) to complete by Inst. of Subtropical Forestry, Chinese Academy of Forestry Sciences.
Anionite-exchange resin D315 used in the present invention, D202, D307, D208, D215, D218, D301 and D354 can be respectively available from Shanghai Huazhen Science and Technology Co., Ltd., Zhejiang Province Zhengguan Industry Co., Ltd.In the present invention, neutral protease used, Sumizyme MP, papoid, bromeline, aspartic protease are all available from Nanning Pang Bo biotechnology company limited.
Embodiment 1
The shark suft bone (water content 8%) that dries is crushed to median size is about 0.1-1mm, accurately take 10kg, adding respectively 0.1kg Sumizyme MP (200,000 U/g) and 100L water, is that 40%NaOH solution is regulated pH to 9.0 with mass concentration, stirs 12h under 55 ℃.Enzymolysis is warming up to 70 ℃ after finishing, insulation 30min, and enzyme goes out.After enzymolysis solution is cooling, filter, get filtrate, with 6mol/L HCl solution, regulate pH value 7, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000), obtain the concentrated solution 9.8L (potentiometric titration detects the content of chondroitin sulfate in concentrated solution) of chondroitin sulfate concentration 20%.With 6mol/L HCl solution, concentrated solution pH is transferred to 2.0, take the flow velocity of 2VB/h, cross the D202 type anion-exchange resin column of wet resin volume as 39L, collect effluent liquid A; Then,, with the flow velocity wash-out of 78L pure water with 1VB/h, collect water lotion B, merge effluent liquid A and water lotion B, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000) is to 10L, spraying drying, obtain collagen polypeptide product 2.35kg, in product, total protein content 92%, yield 23.5%, hydroxyproline content 7.8%, ash content 2.3%, molecular weight is less than 4000 dalton.
Then, anion-exchange resin column is used the flow velocity wash-out of 390L 1.5mol/L NaCl solution with 5VB/h again, collect elutriant, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000) is to 20L, add 60L concentration expressed in percentage by volume 95% ethanol alcohol precipitation, get precipitation, add again 95% ethanol dehydration of 16L concentration expressed in percentage by volume, get solid vacuum-drying, obtain chondroitin sulfate product 1.96kg, chondroitin sulfate cellulose content 97% in product, yield 20.7%, foreign protein content 1.6%.
Shark suft bone raw material and the collagen polypeptide product that makes are carried out the analysis of aminoacid component, acquired results such as following table 1.
Amino acid whose composition analysis result in table 1. shark suft bone raw material and collagen polypeptide
Embodiment 2
The shark suft bone (water content 8%) that dries is crushed to median size is about 0.1-1mm, accurately take 10kg, add respectively 0.15kg bromeline (2400GDU/g) and 150L water, with 6mol/L HCl solution, regulate pH to 6.0, stir 4h under 30 ℃.Enzymolysis is warming up to 70 ℃ after finishing, insulation 60min, and enzyme goes out.After enzymolysis solution is cooling, filter, get filtrate, be 40%NaOH solution adjusting pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000), obtain the concentrated solution 95L of chondroitin sulfate concentration 2%.With 6mol/L HCl solution, concentrated solution pH is transferred to 3.0, take the flow velocity of 0.5VB/h, cross the D315 type anion-exchange resin column of wet resin volume as 57L, collect effluent liquid A; Then,, with the flow velocity wash-out of 114L pure water with 0.5VB/h, collect water lotion B, merge effluent liquid A and water lotion B, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 1000) is to 25L, spraying drying, obtain collagen polypeptide product 2.50kg, in product, total protein content 90%, yield 24.5%, hydroxyproline content 7.6%, ash content 2.5%, molecular weight is less than 4000 dalton.
Then, anion-exchange resin column is used the flow velocity wash-out of 285L 2.5mol/L NaCl solution with 1VB/h again, collect elutriant, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 5000) is to 38L, add 114L concentration expressed in percentage by volume 95% ethanol alcohol precipitation, get precipitation, add again 95% ethanol dehydration of 15L concentration expressed in percentage by volume, get solid vacuum-drying, obtain chondroitin sulfate product 1.90kg, chondroitin sulfate cellulose content 95% in product, yield 19.6%, foreign protein content 1.8%.
Embodiment 3
The shark suft bone (water content 8%) of oven dry is crushed to median size and is about 1-5mm, accurately take 10kg, add respectively 0.05kg aspartic protease (50,000 U/g) and 100L water,, with 6mol/L HCl solution adjusting pH to 2.0, stir 5h under 55 ℃.Enzymolysis is warming up to 80 ℃ after finishing, insulation 10min, and enzyme goes out.After enzymolysis solution is cooling, filter, get filtrate, be 40%NaOH solution adjusting pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000), obtain the concentrated solution 38L of chondroitin sulfate concentration 5%.With 6mol/L HCl solution, concentrated solution pH is transferred to 4.0, take the flow velocity of 1VB/h, cross the D307 type anion-exchange resin column of wet resin volume as 23L, collect effluent liquid A; Then,, with the flow velocity wash-out of 230L pure water with 0.1VB/h, collect water lotion B, merge effluent liquid A and water lotion B, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000) is to 49L, spraying drying, obtain collagen polypeptide product 2.45kg, in product, total protein content 93%, yield 24.8%, hydroxyproline content 7.4%, ash content 2.7%, molecular weight is less than 4000 dalton.
Then, anion-exchange resin column is used the flow velocity wash-out of 23L 6mol/L NaCl solution with 5VB/h again, collect elutriant, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 5000) is to 6.3L, add 19L concentration expressed in percentage by volume 95% ethanol alcohol precipitation, get precipitation, add again 95% ethanol dehydration of 15L concentration expressed in percentage by volume, get solid vacuum-drying, obtain chondroitin sulfate product 1.89kg, chondroitin sulfate cellulose content 94% in product, yield 19.3%, foreign protein content 1.5%.
Embodiment 4
The shark suft bone (water content 8%) of oven dry is crushed to median size and is about 1-5mm, accurately take 10kg, add respectively 0.15kg aspartic protease (50,000 U/g) and 50L water,, with 6mol/L HCl solution adjusting pH to 3.5, stir 6h under 45 ℃.Enzymolysis is warming up to 80 ℃ after finishing, insulation 60min, and enzyme goes out.After enzymolysis solution is cooling, filter, get filtrate, be 40%NaOH solution adjusting pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000), obtain the concentrated solution 13.5L of chondroitin sulfate concentration 15%.With 6mol/L HCl solution, concentrated solution pH is transferred to 5.0, take the flow velocity of 0.1VB/h, cross the D301 type anion-exchange resin column of wet resin volume as 30L, collect effluent liquid A; Then,, with the flow velocity wash-out of 240L pure water with 15VB/h, collect water lotion B, merge effluent liquid A and water lotion B, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000) is to 12L, spraying drying, obtain collagen polypeptide product 2.51kg, in product, total protein content 95%, yield 25.9%, hydroxyproline content 7.6%, ash content 2.5%, molecular weight is less than 4000 dalton.
Then, anion-exchange resin column is used the flow velocity wash-out of 300L 2mol/L NaCl solution with 10VB/h again, collect elutriant, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 6000) is to 10L, add 30L concentration expressed in percentage by volume 95% ethanol alcohol precipitation, get precipitation, add again 95% ethanol dehydration of 16L concentration expressed in percentage by volume, get solid vacuum-drying, obtain chondroitin sulfate product 2.02kg, chondroitin sulfate cellulose content 86% in product, yield 18.9%, foreign protein content 2.2%.
Embodiment 5
The hog snout bone (water content 14%) that dries is crushed to median size is about 5-10mm, accurately take 10kg, add respectively 0.1kg neutral protease (200,000 U/g) and 100L water, stir 10h under 30 ℃.Enzymolysis is warming up to 100 ℃ after finishing, insulation 10min, and enzyme goes out.After enzymolysis solution is cooling, filter, get filtrate, be 40%NaOH solution adjusting pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000), obtain the concentrated solution 13.5L of chondroitin sulfate concentration 10%.Concentrated solution (pH 7.0) is crossed the D354 type anion-exchange resin column of wet resin volume as 25L take the flow velocity of 5VB/h, collect effluent liquid A; Then,, with the flow velocity wash-out of 200L pure water with 0.5VB/h, collect water lotion B, merge effluent liquid A and water lotion B, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000) is to 20L, spraying drying, obtain collagen polypeptide product 3.01kg, in product, total protein content 91%, yield 31.9%, hydroxyproline content 11.4%, ash content 2.4%, molecular weight is less than 4000 dalton.
Then, anion-exchange resin column is used the flow velocity wash-out of 450L 4mol/L NaCl solution with 12VB/h again, collect elutriant, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 6000) is to 27L, add 81L concentration expressed in percentage by volume 95% ethanol alcohol precipitation, get precipitation, add again 95% ethanol dehydration of 11L concentration expressed in percentage by volume, get solid vacuum-drying, obtain chondroitin sulfate product 1.35kg, chondroitin sulfate cellulose content 96% in product, yield 15.1%, foreign protein content 1.7%.
The hog snout bone (water content 14%) that dries is crushed to median size is about 5-10mm, accurately take 10kg, adding respectively 0.01kg Sumizyme MP (200,000 U/g) and 100L water, is that 40%NaOH solution is regulated pH to 10.0 with mass concentration, stirs 5h under 55 ℃.Enzymolysis is warming up to 80 ℃ after finishing, insulation 30min, and enzyme goes out.After enzymolysis solution is cooling, filter, get filtrate,, with 6mol/L HCl solution adjusting pH value 7, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000), obtain the concentrated solution 9L of chondroitin sulfate concentration 14.4%.With 6mol/L HCl solution, concentrated solution pH is transferred to 6.0, take the flow velocity of 10VB/h, cross the D218 type anion-exchange resin column of wet resin volume as 26L, collect effluent liquid A; Then,, with the flow velocity wash-out of 130L pure water with 5VB/h, collect water lotion B, merge effluent liquid A and water lotion B, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000) is to 20L, spraying drying, obtain collagen polypeptide product 3.02kg, in product, total protein content 93%, yield 32.7%, hydroxyproline content 11.8%, ash content 2.3%, molecular weight is less than 4000 dalton.
Then, anion-exchange resin column is used the flow velocity wash-out of 468L 1mol/L NaCl solution with 12VB/h again, collect elutriant, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 5000) is to 13L, add 39L concentration expressed in percentage by volume 95% ethanol alcohol precipitation, get precipitation, add again 95% ethanol dehydration of 10L concentration expressed in percentage by volume, get solid vacuum-drying, obtain chondroitin sulfate product 1.3kg, chondroitin sulfate cellulose content 95% in product, yield 14.4%, foreign protein content 1.8%.
Embodiment 7
The hog snout bone (water content 14%) of oven dry is crushed to median size and is about 1-5mm, accurately take 10kg, add respectively 0.08kg neutral protease (200,000 U/g) and 300L water, stir 2h under 30 ℃.Enzymolysis is warming up to 70 ℃ after finishing, insulation 60min, and enzyme goes out.After enzymolysis solution is cooling, filter, get filtrate, be 40%NaOH solution adjusting pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000), obtain the concentrated solution 64L of chondroitin sulfate concentration 2%.With 6mol/L HCl solution, concentrated solution pH is transferred to 4.0, take the flow velocity of 5VB/h, cross the D315 type anion-exchange resin column of wet resin volume as 23L, collect effluent liquid A; Then,, with the flow velocity wash-out of 46L pure water with 5VB/h, collect water lotion B, merge effluent liquid A and water lotion B, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000) is to 10L, spraying drying, obtain collagen polypeptide product 3.0kg, in product, total protein content 88%, yield 30.7%, hydroxyproline content 11.5%, ash content 2.2%, molecular weight is less than 4000 dalton.
Then, anion-exchange resin column is used the flow velocity wash-out of 460L 2.5mol/L NaCl solution with 15VB/h again, collect elutriant, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 6000) is to 25L, add 75L concentration expressed in percentage by volume 95% ethanol alcohol precipitation, get precipitation, add again 95% ethanol dehydration of 10L concentration expressed in percentage by volume, get solid vacuum-drying, obtain chondroitin sulfate product 1.28kg, chondroitin sulfate cellulose content 93% in product, yield 13.8%, foreign protein content 2.0%.
Embodiment 8
The pig larynx bone (water content 11%) that dries is crushed to median size is about 1-5mm, accurately take 10kg, add respectively 0.12kg papoid (650,000 U/g) and 100L water, with 6mol/L HCl solution, regulate pH to 6.0, stir 10h under 45 ℃.Enzymolysis is warming up to 100 ℃ after finishing, insulation 30min, and enzyme goes out.After enzymolysis solution is cooling, filter, get filtrate, be 40%NaOH solution adjusting pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000), obtain the concentrated solution 26L of chondroitin sulfate concentration 5%.With 6mol/L HCl solution, concentrated solution pH is transferred to 6.0, take the flow velocity of 2VB/h, cross the D215 type anion-exchange resin column of wet resin volume as 33L, collect effluent liquid A; Then,, with the flow velocity wash-out of 3.3L pure water with 1VB/h, collect water lotion B, merge effluent liquid A and water lotion B, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000) is to 15L, spraying drying, obtain collagen polypeptide product 2.96kg, in product, total protein content 89%, yield 29.6%, hydroxyproline content 11.3%, ash content 2.6%, molecular weight is less than 4000 dalton.
Then, anion-exchange resin column is used the flow velocity wash-out of 495L 4mol/LNaCl solution with 0.1VB/h again, collect elutriant, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000) is to 130L, add 390L concentration expressed in percentage by volume 95% ethanol alcohol precipitation, get precipitation, add again 95% ethanol dehydration of 10L concentration expressed in percentage by volume, get solid vacuum-drying, obtain chondroitin sulfate product 1.3kg, chondroitin sulfate cellulose content 90% in product, yield 13.1%, foreign protein content 2.2%.
Embodiment 9
The nose of an ox bone (water content 12%) of oven dry is crushed to median size and is about 5-10mm, accurately take 10kg, add respectively 0.1kg bromeline (2400GDU/g) and 100L water,, with 6mol/L HCl solution adjusting pH to 6.5, stir 5h under 55 ℃.Enzymolysis is warming up to 70 ℃ after finishing, insulation 30min, and enzyme goes out.After enzymolysis solution is cooling, filter, get filtrate, be 40%NaOH solution adjusting pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000), obtain the concentrated solution 27L of chondroitin sulfate concentration 15%.With 6mol/L HCl solution, concentrated solution pH is transferred to 3.0, take the flow velocity of 1VB/h, cross the D202 type anion-exchange resin column of wet resin volume as 73L, collect effluent liquid A; Then,, with the flow velocity wash-out of 730L pure water with 10VB/h, collect water lotion B, merge effluent liquid A and water lotion B, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000) is to 26L, spraying drying, obtain collagen polypeptide product 2.63kg, in product, total protein content 94%, yield 28.1%, hydroxyproline content 10.4%, ash content 2.5%, molecular weight is less than 4000 dalton.
Then, anion-exchange resin column is used the flow velocity wash-out of 365L 1.5mol/L NaCl solution with 0.5VB/h again, collect elutriant, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 8000) is to 40L, add 120L concentration expressed in percentage by volume 95% ethanol alcohol precipitation, get precipitation, add again 95% ethanol dehydration of 32L concentration expressed in percentage by volume, get solid vacuum-drying, obtain chondroitin sulfate product 4.05kg, chondroitin sulfate cellulose content 93% in product, yield 42.8%, foreign protein content 1.9%.
The nose of an ox bone (water content 12%) of oven dry is crushed to median size and is about 5-10mm, accurately take 10kg, add respectively 0.1kg papoid (650,000 U/g) and 100L water,, with 6mol/L HCl solution adjusting pH to 6.5, stir 6h under 55 ℃.Enzymolysis is warming up to 100 ℃ after finishing, insulation 10min, and enzyme goes out.After enzymolysis solution is cooling, filter, get filtrate, be 40%NaOH solution adjusting pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000), obtain the concentrated solution 43L of chondroitin sulfate concentration 10%.With 6mol/L HCl solution, concentrated solution pH is transferred to 5.0, take the flow velocity of 0.5VB/h, cross the D215 type anion-exchange resin column of wet resin volume as 108L, collect effluent liquid A; Then,, with the flow velocity wash-out of 108L pure water with 12VB/h, collect water lotion B, merge effluent liquid A and water lotion B, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000) is to 13L, spraying drying, obtain collagen polypeptide product 2.62kg, in product, total protein content 95%, yield 28.3%, hydroxyproline content 10.2%, ash content 2.2%, molecular weight is less than 4000 dalton.
Then, anion-exchange resin column is used the flow velocity wash-out of 324L 3mol/L NaCl solution with 1VB/h again, collect elutriant, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 5000) is to 86L, add 258L concentration expressed in percentage by volume 95% ethanol alcohol precipitation, get precipitation, add again 95% ethanol dehydration of 34L concentration expressed in percentage by volume, get solid vacuum-drying, obtain chondroitin sulfate product 4.31kg, chondroitin sulfate cellulose content 97% in product, yield 47.5%, foreign protein content 2.3%.
Embodiment 11
The pig larynx bone (water content 11%) that dries is crushed to median size is about 1-5mm, accurately take 10kg, add respectively 0.08kg neutral protease (200,000 U/g) and 200L water, stir 12h under 55 ℃.Enzymolysis is warming up to 70 ℃ after finishing, insulation 30min, and enzyme goes out.After enzymolysis solution is cooling, filter, get filtrate, be 40%NaOH solution adjusting pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 3000), obtain the concentrated solution 135L of chondroitin sulfate concentration 1%.With 6mol/L HCl solution, concentrated solution pH is transferred to 4.0, take the flow velocity of 8VB/h, cross the D208 type anion-exchange resin column of wet resin volume as 13.5L, collect effluent liquid A; Then,, with the flow velocity wash-out of 54L pure water with 5VB/h, collect water lotion B, merge effluent liquid A and water lotion B, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 1000) is to 13L, spraying drying, obtain collagen polypeptide product 3.18kg, in product, total protein content 92%, yield 32.9%, hydroxyproline content 11.2%, ash content 2.0%, molecular weight is less than 4000 dalton.
Then, anion-exchange resin column is used the flow velocity wash-out of 67.5L 3mol/L NaCl solution with 12VB/h again, collect elutriant, be that 40%NaOH solution is regulated pH value 7 with mass concentration, ultrafiltration and concentration (the ultra-filtration membrane molecular weight cut-off is 6000) is to 27L, add 81L concentration expressed in percentage by volume 95% ethanol alcohol precipitation, get precipitation, add again 95% ethanol dehydration of 10L concentration expressed in percentage by volume, get solid vacuum-drying, obtain chondroitin sulfate product 1.35kg, chondroitin sulfate cellulose content 96% in product, yield 14.6%, foreign protein content 1.5%.
Claims (9)
1. method of utilizing animal cartilage to prepare chondroitin sulfate and collagen polypeptide, it is characterized in that described method is: the animal cartilage raw material pulverizing is obtained cartilage powder, cartilage powder with protease hydrolysis after ultrafiltration and concentration obtain concentrated solution, concentrated solution is crossed anion-exchange resin column, collect effluent liquid A, then use the pure water wash-out, collect water lotion B, ultrafiltration and concentration after effluent liquid A and water lotion B merge, then spraying drying makes collagen polypeptide; Anion-exchange resin column is used the NaCl eluant solution, the elutriant ultrafiltration and concentration makes chondroitin sulfate by alcohol precipitation, dehydration, drying again;
Said method comprising the steps of:
(1) be 0.1-10mm with animal cartilage raw material pulverizing to median size, obtain cartilage powder; Cartilage powder adds proteolytic enzyme and water, regulates the extremely optimum pH of described proteolytic enzyme of pH value, carries out enzyme digestion reaction, the controlled enzymatic hydrolysis temperature between 30-55 ℃, stirring reaction 2-12h; Then heat the enzyme that goes out, cooled and filtered is got filtrate, and adjust pH is neutral, and adopting molecular weight cut-off is that the daltonian ultra-filtration membrane ultrafiltration and concentration of 1000-8000 obtains concentrated solution, and in described concentrated solution, the mass percentage concentration of chondroitin sulfate is 1～20%; Described proteolytic enzyme is one of following: neutral protease, Sumizyme MP, papoid, bromeline or aspartic protease; The consumption of described proteolytic enzyme is the 0.1-1.5% of cartilage powder quality; The consumption of water is cartilage powder quality 5-30 times;
(2) step (1) gained concentrated solution adjust pH, to 2.0-7.0, is crossed anion-exchange resin column, collects effluent liquid A; Use the pure water wash-out, collect water lotion B, effluent liquid A and water lotion B merge, and regulating pH value is neutrality, the employing molecular weight cut-off be the daltonian ultra-filtration membrane ultrafiltration and concentration of 1000-8000 to the mass concentration of albumen be 5-30%, then spraying drying obtains collagen polypeptide;
(3) anion-exchange resin column of step (2) is eluent with 1-6mol/L NaCl solution again, chondroitin sulfate is carried out wash-out, collect elutriant, it is neutral regulating the pH value, to adopt molecular weight cut-off be the daltonian ultra-filtration membrane ultrafiltration and concentration of 1000-8000 to the mass concentration of chondroitin sulfate be 1-30%, through alcohol precipitation, dehydration, drying, make chondroitin sulfate.
2. the method for claim 1, it is characterized in that described animal cartilage raw material be after drying or drying water content lower than the animal cartilage of 15wt%.
3. the method for claim 1, is characterized in that described anionite-exchange resin is one of following: D315, D202, D307, D208, D215, D218, D301 or D354.
4. the method for claim 1, is characterized in that in described step (2), the wet resin volume of described anionite-exchange resin is counted 10-30L/kg with the quality of chondroitin sulfate in concentrated solution.
5. the method for claim 1, is characterized in that in described step (2), and described concentrated solution adjust pH, to 2.0-7.0, is crossed anion-exchange resin column, and the described flow velocity of crossing anionite-exchange resin is 0.1-10VB/h.
6. the method for claim 1, is characterized in that using the pure water wash-out in described step (2), and described pure water is 0.1-15VB/h by the flow velocity of anionite-exchange resin, and the consumption of described pure water is 0.1-10 times of column volume.
7. the method for claim 1, is characterized in that in described step (3), and the consumption of NaCl solution is 1-20 times of column volume, and the flow velocity by anionite-exchange resin is 0.1-15VB/h.
8. the method for claim 1, is characterized in that in described step (1), and the described heating enzyme that goes out is at 70-100 ℃, and under the condition of insulation 10-60min, enzyme goes out.
9. the method for claim 1, it is characterized in that in described step (3), described alcohol precipitation, dehydration, drying operate by the following method: adding while stirring volume is concentration expressed in percentage by volume 95% aqueous ethanolic solution of 3～4 times of the concentrated solution volume after ultrafiltration and concentration, get precipitation, add again concentration expressed in percentage by volume 95% aqueous ethanolic solution dehydration, finally, vacuum-drying obtains chondroitin sulfate.
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| CN104450841A (en) * | 2013-06-27 | 2015-03-25 | 青岛贝尔特生物科技有限公司 | Method for producing chondroitin sulfate and co-producing hydrolyzed collagen from fish cartilage |
| CN104450842A (en) * | 2013-06-27 | 2015-03-25 | 青岛贝尔特生物科技有限公司 | Method for producing chondroitin sulfate and co-producing hydrolyzed collagen from fish cartilage |
| CN104911240A (en) * | 2014-03-14 | 2015-09-16 | 嘉兴纽迪康生物科技有限公司 | Process for co-production of collagen calcium and polypeptide in chondroitin sulfate production |
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| CN102093490A (en) * | 2010-12-27 | 2011-06-15 | 浙江工业大学 | Method for preparing high-purity chondroitin sulfate |
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