CN102093490B - Method for preparing high-purity chondroitin sulfate - Google Patents
Method for preparing high-purity chondroitin sulfate Download PDFInfo
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Abstract
The invention discloses a method for preparing chondroitin sulfate, which comprises the following steps: boiling animal cartilage, performing enzymolysis and filtering to obtain enzymolysis liquid; subjecting the enzymolysis liquid to ultrafiltration concentration to obtain primary concentrate; passing the primary concentrate through cation exchange resin, and collecting effluent; washing the cation exchange resin with water, and collecting and mixing effluent; subjecting the effluent to ultrafiltration concentration to obtain secondary concentrate; and precipitating the secondary concentrate in ethanol, dewatering and drying to obtain chondroitin sulfate. The method disclosed by the invention has the advantages of high selectivity, high product purity, high yield, low energy consumption, low ethanol consumption, mild process conditions, simple operation and the like.
Description
(1) technical field
The present invention relates to a kind of method for preparing the high purity CHS, be specifically related to a kind of directly method of separation and purification CHS from the cartilage enzymolysis solution.
(2) background technology
(Chondroitin Sulfate CS) is the one type of acidic mucopolysaccharide that is distributed widely in the animal cartilage tissue to CHS.The polysaccharide that its molecular structure is formed for the repetition disaccharide unit that is made up of D-glucuronic acid and N-acetylamino galactosamine.Be mainly used in clinically and prevent and treat neurodynia, nervous migraine, sacroiliitis, omarthralgia, abdominal postoperative pain, coronary heart disease, stenocardia and myocardial infarction etc., the prevention of diseases such as the hypoacusia that dysacousis that causes for Streptomycin sulphate and various noise cause, tinnitus and treatment also have good curative effect.
China is a large agricultural country, and livestock industry is flourishing, and the raw material sources of production CHS are extensive, kind abundant.Traditional production process mainly adopts enzymolysis process or alkali to carry enzymolysis process and extracts CHS, obtains the CHS bullion through ethanol sedimentation.Then, adopt oxidizing water solution (Zhang Zhibin, the new enzymolysis process technology of CHS production, 2010 again; 98), ethanol precipitation method (Liu Anjun etc., the extraction and the chemical composition analysis thereof of ox trunnion cartilage polysaccharide, 2,009 27 (4):; 25 (11): 1315-1319), (Chen Danqing, swamp eel bone CHS extract the preliminary study of purifying, 2008 to isoelectric point method; 20:1075-1079,1034), resin adsorption method (Zhang Guozhi, 200810000635.6) etc. carries out purifying to bullion; At last, through the alcohol precipitation second time, obtain CHS.Wherein, first three process for purification complex operation, product yield is lower, and the content of foreign protein is higher in the product; Though and the resin adsorption method quality product is better, but still be raw material, exist the ethanol consumption big, the more high shortcoming of production cost with the CHS bullion.Song Jiabin etc. then adopt strongly basic anion exchange resin separation and purification CHS (200510085564.0) from enzymolysis solution, because CHS molecular weight big (10000-50000D) requires polymeric adsorbent to have special macroporous structure; Selectivity is relatively poor, and wash-out is difficulty also, therefore; Product gas purity and yield are all not high; And the NaCl consumption that wash-out uses is big, pollutes more seriously, is inappropriate for industrialized production.
Ion exchange technique is to utilize the difference of binding ability between ionite and the different ions, and on exchanger, and the material that can not be adsorbed is directly eluted with some ionic adsorption in the solution, thus the purpose that reaches separation, purifies.Ion exchange resin nontoxicity and can using repeatedly, this method is used less or without organic solvent, is had advantages such as selectivity is good, equipment is simple, easy to operate.Ultra-filtration technique is to realize the separation to large and small molecule, concentrated and purification through the microvoid structure on film surface.Gentle, the no composition of operational condition destroys, energy consumption is low, easy and simple to handle.More than two kinds of technology be widely used in the separation of bioactive ingredients.
Adopt Zeo-karb, and combine ultrafiltration can realize direct separation purification of sulphuric acids chrondroitin from the cartilage enzymolysis solution,, can obtain highly purified CHS through a step alcohol precipitation.This method is not also appeared in the newspapers at present, is a kind of novel method of separation and purification CHS, will help promoting the transformation of traditional processing technology, and promotes the sustainable development of China's animal extracts industry.
(3) summary of the invention
The objective of the invention is to solve that the amount of ethanol that exists in traditional production technique of chondroitin sulfate is big, product purity is not high, foreign protein content is than problems such as height, and a kind of high yield, less energy-consumption, high purity preparing chondroitin sulfates easy and simple to handle are provided.
For realizing the foregoing invention purpose, the present invention specifically adopts following technical scheme:
A kind of preparing chondroitin sulfates comprises the steps: animal cartilage is boiled soft, enzymolysis, filters to obtain enzymolysis solution; Enzymolysis solution obtains once concentration liquid through ultrafiltration and concentration; Once concentration liquid is passed through Zeo-karb; Collect effluent, use the water washing Zeo-karb then, collect and the merging effluent; Effluent obtains secondary concentration liquid through ultrafiltration and concentration, and secondary concentration liquid is through alcohol precipitation, dehydration, the dry CHS that gets.
One of further, it is one of following that Zeo-karb of the present invention is selected from: DK110, and D113,732, HD-8, HZ-016, D001, D061, D152, PCR642Ca or C150s, preferred following: DK110, D113, HD-8, PCR642Ca or C150s.
Further, the volume of described Zeo-karb is preferably 1～5 times for long-pending 0.1～10 times of once concentration liquid.Described once concentration liquid is 0.1～10BV/h through the flow velocity of Zeo-karb, is preferably 1～6BV/h.
Further, described water is 0.1～15BV/h through the flow velocity of Zeo-karb, is preferably 1～10BV/h; Institute's water requirement is 2～10 times a Zeo-karb volume.
Further, described enzymolysis solution is 1～20% through the mass concentration of ultrafiltration and concentration to CHS, is preferably 5～15%; The molecular weight cut-off of used ultra-filtration membrane is 3000-8000.
Further, described effluent is 1～30% through the mass concentration of ultrafiltration and concentration to CHS, is preferably 5～20%; The molecular weight cut-off of used ultra-filtration membrane is 1000-3000.
Further, in the described alcohol precipitation, preferably using volume(tric)fraction is 95% ethanol, and volume(tric)fraction is 95% ethanol and the mixed volume ratio of secondary concentration liquid is 2～10: 1, is preferably 2～5: 1.
The enzyme that uses in the enzymolysis process of the present invention is trypsinase.
Hollow fiber membrane ultrafiltration device and rolling ultra-fine filter are adopted in ultrafiltration of the present invention.
The present invention is concrete to recommend described preparing chondroitin sulfates to carry out according to following steps: with animal cartilage boil soft after, use trypsin digestion, filtration obtains enzymolysis solution; Using molecular weight cut-off is 1～20% as the ultra-filtration membrane of 3000-8000 with the mass concentration of enzymolysis solution ultrafiltration and concentration to CHS; The once concentration liquid of gained is passed through Zeo-karb with the flow velocity of 0.1～10BV/h; Collect effluent, the volume of described Zeo-karb is long-pending 0.1～10 times of once concentration liquid; Use 2～10 times then to the water of Zeo-karb volume flow velocity washing resin, collect and also merge effluent with 0.1～15BV/h; Using molecular weight cut-off is 1～30% as the ultra-filtration membrane of 1000-3000 with the mass concentration of effluent ultrafiltration and concentration to CHS; Get secondary concentration liquid, the ethanol that adds 2～10 times long-pending volume(tric)fraction 95% of secondary concentration liquid again carries out getting CHS after alcohol precipitation, dehydration, the drying; Described resin cation(R.C.) is one of following: DK110, D113, HD-8,732, HZ-016, D001, D061, D152, PCR642Ca or C150s.
The present invention adopts Zeo-karb, and combines ultra-filtration technique high efficiency separation CHS, and compared with prior art, its beneficial effect is embodied in:
(1) can realize from the cartilage enzymolysis solution directly high efficiency separation CHS, substitute in the traditional technology ethanol sedimentation Crude polysaccharides and oxydrolysis or the proteic production process of adsorption-edulcoration, simplify Production Flow Chart, cut down the ethanol consumption greatly.
(2) processing condition are gentle, effectively improved product gas purity and yield.For example: with pig larynx bone is raw material, adopts the inventive method to prepare CHS, with enzymolysis-oxidizing water solution (Zhang Zhibin; 2010) compare; Can make product content be increased to 98.5% from 98.2%, foreign protein content is reduced to 2.1% from 2.6%, and yield is increased to 17.5% from 16.6%.With the hog snout bone is raw material, adopts the inventive method to prepare CHS, compares with enzymolysis-anion-exchange resin method (Song Jiabin etc., 200510085564.0), can make product content be increased to 99% from 87.5%, and foreign protein content is merely 2.0%.
(3) resin decolorization is respond well, has got rid of the use of discoloring agents such as hydrogen peroxide, and product is safer.
(4) energy consumption is low, and has reduced production cost, and is simple to operate, is convenient to large-scale industrial production.
To sum up, the inventive method have that selectivity is good, product purity and yield is high, energy consumption and the ethanol consumption is low, processing condition are gentle, simple operation and other advantages.CHS can be widely used in fields such as medicine, foods and cosmetics, has better market prospect.
(4) embodiment
Below with specific embodiment technical scheme of the present invention is described, but protection scope of the present invention is not limited thereto.
The testing conditions of CHS described in following examples and method of calculation are:
The qualitative and quantitative analysis of product: with commercially available CHS standard substance is contrast, carries out efficient liquid phase chromatographic analysis, appearance time and standard substance basically identical.The quality of CHS is to calculate with the typical curve (relation between peak area and the concentration) that the CHS standard substance are drawn in the product.
Quality * 100 of the quality/animal cartilage of CHS in the yield of CHS (%)=product in the product described in following examples; Quality/the quality product (dry product) * 100 of CHS in the content of CHS (%)=product in the said product; Proteinic quality/quality product (dry product) * 100 in the content of foreign protein (%)=product in the said product.
Embodiment 1
1) get 20Kg hog snout bone (water cut 12%), add 400L water, 4KgNaCl is warming up to 100 ℃, insulation 2h; Naturally cool to 45 ℃-48 ℃, the use mass concentration is 40% NaOH solution adjusting pH to 9.0, adds 300g trypsin digestion 6h, and enzymolysis is warming up to 70 ℃ after finishing, and stirs 30min, filters, and gets the 400L enzymolysis solution;
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 3000, gets the 68L liquid concentrator;
3) be the DK110 type Zeo-karb of 68L with the flow velocity of 1.0BV/h through volume with the 68L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 272L water with 2.0BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 3000, gets 34L secondary concentration liquid;
5) add the ethanol of 102L 95% in the 34L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 3.38Kg.Through detecting, the content of CHS is 97% in the product, and the content of foreign protein is 2.5%, and yield is 16.4%.
Embodiment 2
1) with embodiment 1
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 5000, gets the 33L liquid concentrator;
3) be the HD-8 type Zeo-karb of 66L with the flow velocity of 2.0BV/h through volume with the 33L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 132L water with 3.0BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 2000, gets 22L secondary concentration liquid;
5) add the ethanol of 66L 95% in the 22L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 3.30Kg.Through detecting, the content of CHS is 97% in the product, and the content of foreign protein is 2.2%, and yield is 16.0%.
Embodiment 3
1) with embodiment 1
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 6000, gets the 41L liquid concentrator;
3) be the C150s type Zeo-karb of 123L with the flow velocity of 0.5BV/h through volume with the 41L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 492L water with 0.1BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 3000, gets 22L secondary concentration liquid;
5) add the ethanol of 110L 95% in the 22L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 3.27Kg.Through detecting, the content of CHS is 99% in the product, and the content of foreign protein is 2.0%, and yield is 16.2%.
Embodiment 4
1) with embodiment 1
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 3000, gets the 43L liquid concentrator;
3) be the HZ-016 type Zeo-karb of 22L with the flow velocity of 6BV/h through volume with the 43L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 132L water with 5BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 1000, gets 35L secondary concentration liquid;
5) add the ethanol of 280L 95% in the 35L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 3.48Kg.Through detecting, the content of CHS is 95% in the product, and the content of foreign protein is 2.8%, and yield is 16.5%.
Embodiment 5
1) with embodiment 1
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 5000, gets the 172L liquid concentrator;
3) be the D001 type Zeo-karb of 344L with the flow velocity of 8BV/h through volume with the 172L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 1376L water with 10BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 2000, gets 69L secondary concentration liquid;
5) add the ethanol of 345L 95% in the 69L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 3.44Kg.Through detecting, the content of CHS is 94% in the product, and the content of foreign protein is 2.9%, and yield is 16.2%.
Embodiment 6
1) with embodiment 1
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 7000, gets the 68L liquid concentrator;
3) be the D061 type Zeo-karb of 204L with the flow velocity of 1.0BV/h through volume with the 68L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 408L water with 1.0BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 3000, gets 23L secondary concentration liquid;
5) add the ethanol of 69L 95% in the 23L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 3.41Kg.Through detecting, the content of CHS is 94% in the product, and the content of foreign protein is 2.5%, and yield is 16.1%.
Embodiment 7
1) with embodiment 1
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 6000, gets the 183L liquid concentrator;
3) be the 732 type Zeo-karbs of 18L with the flow velocity of 10BV/h through volume with the 183L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 146L water with 15BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 1000, gets 12L secondary concentration liquid;
5) add the ethanol of 120L 95% in the 12L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 3.65Kg.Through detecting, the content of CHS is 92% in the product, and the content of foreign protein is 3.1%, and yield is 16.8%.
Embodiment 8
1) get 20Kg pig larynx bone (water cut 10%), add 400L water, 4KgNaCl is warming up to 100 ℃, insulation 2h; Naturally cool to 45 ℃-48 ℃, the use mass concentration is 40% NaOH solution adjusting pH to 9.0, adds 300g trypsin digestion 6h, and enzymolysis is warming up to 70 ℃ after finishing, and stirs 30min, filters, and gets the 400L enzymolysis solution;
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 7000, gets the 24L liquid concentrator;
3) be the D152 type Zeo-karb of 120L with the flow velocity of 2.0BV/h through volume with the 24L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 720L water with 1.0BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 2000, gets 71L secondary concentration liquid;
5) add the ethanol of 284L 95% in the 71L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 3.55Kg.Through detecting, the content of CHS is 98.5% in the product, and the content of foreign protein is 2.1%, and yield is 17.5%.
Embodiment 9
1) get 20Kg shark bone (water cut 7%), add 400L water, 4KgNaCl is warming up to 100 ℃, insulation 2h; Naturally cool to 45 ℃-48 ℃, the use mass concentration is 40% NaOH solution adjusting pH to 9.0, adds 300g trypsin digestion 6h, and enzymolysis is warming up to 70 ℃ after finishing, and stirs 30min, filters, and gets the 400L enzymolysis solution;
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 8000, gets the 40L liquid concentrator;
3) be the HD-8 type Zeo-karb of 120L with the flow velocity of 0.5BV/h through volume with the 40L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 1200L water with 2.0BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 3000, gets 60L secondary concentration liquid;
5) add the ethanol of 240L 95% in the 60L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 5.96Kg.Through detecting, the content of CHS is 99% in the product, and the content of foreign protein is 1.2%, and yield is 29.5%.
Embodiment 10
1) with embodiment 9
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 5000, gets the 77L liquid concentrator;
3) be the PCR642Ca type Zeo-karb of 77L with the flow velocity of 2.0BV/h through volume with the 77L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 154L water with 3.0BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 2000, gets 31L secondary concentration liquid;
5) add the ethanol of 124L 95% in the 31L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 6.12Kg.Through detecting, the content of CHS is 97% in the product, and the content of foreign protein is 1.7%, and yield is 29.7%.
Embodiment 11
1) with embodiment 9
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 6000, gets the 31L liquid concentrator;
3) be the C150s type Zeo-karb of 310L with the flow velocity of 1.0BV/h through volume with the 31L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 1240L water with 5.0BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 1000, gets 31L secondary concentration liquid;
5) add the ethanol of 248L 95% in the 31L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 6.19Kg.Through detecting, the content of CHS is 97% in the product, and the content of foreign protein is 1.8%, and yield is 30.0%.
Embodiment 12
1) get the self-conceit pipe of 20Kg (water cut 18%), add 400L water, 4KgNaCl is warming up to 100 ℃, insulation 2h; Naturally cool to 45 ℃-48 ℃, the use mass concentration is 40% NaOH solution adjusting pH to 9.0, adds 300g trypsin digestion 6h, and enzymolysis is warming up to 70 ℃ after finishing, and stirs 30min, filters, and gets the 400L enzymolysis solution;
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 8000, gets the 51L liquid concentrator;
3) be the PCR642Ca type Zeo-karb of 255L with the flow velocity of 0.1BV/h through volume with the 51L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 1530L water with 0.5BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 2000, gets 51L secondary concentration liquid;
5) add the ethanol of 153L 95% in the 51L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 2.57Kg.Through detecting, the content of CHS is 94% in the product, and the content of foreign protein is 2.4%, and yield is 12.1%.
Embodiment 13
1) get 20Kg nose of an ox bone (water cut 25%), add 400L water, 4KgNaCl is warming up to 100 ℃, insulation 2h; Naturally cool to 45 ℃-48 ℃, the use mass concentration is 40% NaOH solution adjusting pH to 9.0, adds 300g trypsin digestion 6h, and enzymolysis is warming up to 70 ℃ after finishing, and stirs 30min, filters, and gets the 400L enzymolysis solution;
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 7000, gets the 42L liquid concentrator;
3) be the C150s type Zeo-karb of 336L with the flow velocity of 2.0BV/h through volume with the 42L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 1344L water with 2.0BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 2000, gets 33L secondary concentration liquid;
5) add the ethanol of 66L 95% in the 33L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 3.33Kg.Through detecting, the content of CHS is 98% in the product, and the content of foreign protein is 2.2%, and yield is 16.3%.
Embodiment 14
1) with embodiment 13
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 3000, gets the 34L liquid concentrator;
3) be the D113 type Zeo-karb of 68L with the flow velocity of 6.0BV/h through volume with the 34L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 680L water with 3.0BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 1000, gets 34L secondary concentration liquid;
5) add the ethanol of 102L 95% in the 34L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 3.41Kg.Through detecting, the content of CHS is 98% in the product, and the content of foreign protein is 2.2%, and yield is 16.7%.
Embodiment 15
1) get 20Kg pigeon breast bone (water cut 43%), add 400L water, 4KgNaCl is warming up to 100 ℃, insulation 2h; Naturally cool to 45 ℃-48 ℃, the use mass concentration is 40% NaOH solution adjusting pH to 9.0, adds 300g trypsin digestion 6h, and enzymolysis is warming up to 70 ℃ after finishing, and stirs 30min, filters, and gets the 400L enzymolysis solution;
2) enzymolysis solution is cooled to 40 ℃ naturally, regulates pH to 7.0-8.0 with the hydrochloric acid soln of 6mol/L, under WP 0.15MPa, carry out ultrafiltration and concentration with hollow fiber membrane ultrafiltration device, molecular weight cut-off is 5000, gets the 206L liquid concentrator;
3) be the DK110 type Zeo-karb of 206L with the flow velocity of 2.0BV/h through volume with the 206L liquid concentrator, collect effluent; Then with the flow velocity washing Zeo-karb of 1236L water with 2.0BV/h;
4) merge effluent, the use mass concentration is 40% NaOH solution adjusting pH to 7.0-8.0, under WP 0.5MPa, carries out ultrafiltration and concentration with the rolling ultra-fine filter, and molecular weight cut-off is 3000, gets 206L secondary concentration liquid;
5) add the ethanol of 412L 95% in the 206L secondary concentration liquid, alcohol precipitation while stirring, then, and with twice, 80 ℃ of vacuum-drying 4h of ethanol dehydration of 20L95%, must CHS product 2.06Kg.Through detecting, the content of CHS is 98% in the product, and the content of foreign protein is 1.9%, and yield is 10.1%.
Claims (9)
1. a preparing chondroitin sulfates comprises the steps: animal cartilage is boiled soft, enzymolysis, filters to obtain enzymolysis solution; Enzymolysis solution obtains once concentration liquid through ultrafiltration and concentration; Once concentration liquid is passed through Zeo-karb; Collect effluent, use the water washing Zeo-karb then, collect and the merging effluent; Effluent obtains secondary concentration liquid through ultrafiltration and concentration, and secondary concentration liquid is through alcohol precipitation, dehydration, the dry CHS that gets.
2. preparing chondroitin sulfates as claimed in claim 1 is characterized in that described Zeo-karb is one of following: DK110, D113,732, HD-8, HZ-016, D001, D061, D152, PCR642Ca or C150s.
3. according to claim 1 or claim 2 preparing chondroitin sulfates, the volume that it is characterized in that described Zeo-karb is long-pending 0.1 ~ 10 times of once concentration liquid.
4. preparing chondroitin sulfates as claimed in claim 3 is characterized in that described once concentration liquid is 0.1 ~ 10BV/h through the flow velocity of Zeo-karb.
5. preparing chondroitin sulfates as claimed in claim 1 is characterized in that described water is 0.1 ~ 15BV/h through the flow velocity of Zeo-karb; Institute's water requirement is 2 ~ 10 times of Zeo-karb volumes.
6. preparing chondroitin sulfates as claimed in claim 1 is characterized in that described enzymolysis solution is 1 ~ 20% through the mass concentration of ultrafiltration and concentration to CHS; The molecular weight cut-off of used ultra-filtration membrane is 3000-8000.
7. preparing chondroitin sulfates as claimed in claim 1 is characterized in that described effluent is 1 ~ 30% through the mass concentration of ultrafiltration and concentration to CHS; The molecular weight cut-off of used ultra-filtration membrane is 1000-3000.
8. preparing chondroitin sulfates as claimed in claim 1 is characterized in that in the described alcohol precipitation, and volume(tric)fraction is 95% ethanol and the mixed volume ratio of secondary concentration liquid is 2 ~ 10:1.
9. preparing chondroitin sulfates as claimed in claim 1 is characterized in that described method carries out according to following steps: with animal cartilage boil soft after, use trypsin digestion, filter and obtain enzymolysis solution; Using molecular weight cut-off is 1 ~ 20% as the ultra-filtration membrane of 3000-8000 with the mass concentration of enzymolysis solution ultrafiltration and concentration to CHS; The once concentration liquid of gained is passed through Zeo-karb with the flow velocity of 0.1 ~ 10BV/h; Collect effluent, the volume of described Zeo-karb is long-pending 0.1 ~ 10 times of once concentration liquid; Use 2 ~ 10 times then to the water of Zeo-karb volume flow velocity washing resin, collect and also merge effluent with 0.1 ~ 15BV/h; Using molecular weight cut-off is 1 ~ 30% as the ultra-filtration membrane of 1000-3000 with the mass concentration of effluent ultrafiltration and concentration to CHS; Get secondary concentration liquid, the ethanol that adds 2 ~ 10 times long-pending volume(tric)fraction 95% of secondary concentration liquid again carries out getting CHS after alcohol precipitation, dehydration, the drying; Described resin cation(R.C.) is one of following: DK110, D113, HD-8,732, HZ-016, D001, D061, D152, PCR642Ca or C150s.
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| CN102838691A (en) * | 2012-08-29 | 2012-12-26 | 宁波绿之健药业有限公司 | Extracting and purifying method of chondroitin sulfate |
| CN103408675A (en) * | 2012-12-26 | 2013-11-27 | 嘉兴恒杰生物制药有限公司 | Organic-solvent-free extraction process of chondroitin sulfate |
| CN103172764B (en) * | 2013-04-01 | 2015-05-20 | 管桂义 | Method for producing chondroitin by taking duck tracheas as raw material |
| CN103724453B (en) * | 2013-11-26 | 2015-12-02 | 沃太能源南通有限公司 | A kind of extraction process of shark cartilage |
| CN110713557B (en) * | 2019-12-10 | 2021-10-26 | 无棣县兴亚生物科技有限公司 | Process for extracting chondroitin by using meat and bone meal waste pulp through biological method |
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