CN114457138A - Method for removing type II collagen peptide polysaccharide - Google Patents
Method for removing type II collagen peptide polysaccharide Download PDFInfo
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- 239000005017 polysaccharide Substances 0.000 title claims abstract description 25
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- 108010041390 Collagen Type II Proteins 0.000 title claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 42
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- 238000006731 degradation reaction Methods 0.000 claims abstract description 16
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- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 12
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 12
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- 102000057297 Pepsin A Human genes 0.000 claims description 5
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- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 claims 7
- 108010013639 Peptidoglycan Proteins 0.000 claims 7
- 238000002525 ultrasonication Methods 0.000 claims 1
- 238000000926 separation method Methods 0.000 abstract description 14
- 238000005516 engineering process Methods 0.000 abstract description 7
- 102000008186 Collagen Human genes 0.000 abstract description 6
- 108010035532 Collagen Proteins 0.000 abstract description 6
- 229920001436 collagen Polymers 0.000 abstract description 6
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- 241000272517 Anseriformes Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 229920002567 Chondroitin Polymers 0.000 description 1
- 102000016942 Elastin Human genes 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 206010057040 Temperature intolerance Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
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- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 description 1
- -1 chondroitin sulfate Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 230000003631 expected effect Effects 0.000 description 1
- 230000008543 heat sensitivity Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000001728 nano-filtration Methods 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
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- 238000003307 slaughter Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
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- 229930003231 vitamin Natural products 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
A method for removing type II collagen peptide polysaccharide comprises the following steps: (1) pretreating raw materials; (2) preparing an enzymolysis liquid; (3) degrading polysaccharide; (4) and (4) ultrafiltration desugarization. The method prepares the type II collagen peptide with low polysaccharide content by combining an ultrasonic degradation technology and an ultrafiltration membrane separation technology, and has the advantages of good desugaring effect, high desugaring efficiency, low loss rate of the collagen peptide, no organic solvent residue, environmental protection and the like.
Description
Technical Field
The invention belongs to the technical field of biological extraction, and particularly relates to a method for removing II type collagen peptide polysaccharide.
Background
China owns the largest number of domestic animals in the world, such as pigs, cattle, sheep, chickens and ducks. As the total production of meat increases, the livestock slaughter waste also increases. Nearly 1000 million tons of various livestock bones are produced every year in China, which is undoubtedly a huge resource. However, in the current consumer market, most bones other than the spareribs and the bones of the body cavities can be directly used for diet, and the demand is large, and the majority of the bones are not fully utilized. The animal bone is rich in various mineral substances such as protein, amino acid, chondroitin, vitamins and the like, and the content of calcium, phosphorus, iron and the like is high. Animal bones, particularly animal cartilage, are rich in nutrients, and therefore, in recent years, extraction of type II collagen peptides from animal cartilage has become a hot spot.
However, in the process of treating cartilage, the enzymatic hydrolysate after primary enzymolysis contains a large amount of polysaccharides such as chondroitin sulfate, and currently, the common methods for separating the polysaccharides mainly include fractional precipitation and chromatographic separation. The fractional precipitation method mainly utilizes the fact that polysaccharide has low solubility in ethanol, and polysaccharide molecules with different polymerization degrees are precipitated and separated out respectively along with the gradual increase of the volume fraction of ethanol. However, in the fractional precipitation method, some polypeptide molecules are precipitated as the volume fraction of alcohol increases. The chromatographic separation method mainly comprises gel column chromatography and ion exchange chromatography. Gel column chromatography mainly utilizes selective permeation of molecules with different sizes in a porous stationary phase to achieve separation; ion exchange chromatography provides separation based on the affinity of the constituent ions for the resin. These conventional methods are cumbersome to operate and have high operating costs.
Compared with two methods of fractional precipitation and chromatographic separation, the membrane separation method has the advantages that the treatment conditions are mild and safe, no other reagent is mixed into the membrane separation method, the membrane separation method is cleaner and pollution-free, nutrient components cannot be damaged by the ultrafiltration membrane, and the polysaccharide in the enzymatic hydrolysate is separated without additional treatment. Application publication No. CN111808185A discloses a method for extracting elastin peptide from bovine cartilage, which comprises high-temperature extraction, and then separating with permeable membrane and nanofiltration membrane to obtain protein peptide. The method adopts two membrane separation technologies, so that the cost is high, and the separation efficiency is low.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a method for removing II type collagen peptide polysaccharide, which prepares the II type collagen peptide with low polysaccharide content by combining an ultrasonic degradation technology and an ultrafiltration membrane separation technology and has the advantages of good desugaring effect, high desugaring efficiency, low protein peptide loss rate, no organic solvent residue and environmental protection.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for removing type II collagen peptide polysaccharide comprises the following steps:
(1) pretreatment of raw materials: selecting animal cartilage from which chondroitin sulfate is extracted, crushing, and then carrying out degreasing, impurity protein removal and decalcification treatment;
(2) preparing enzymolysis liquid: adding protease into the pretreated animal cartilage in the step (1) for enzymolysis for 2-6h, inactivating the enzyme, and centrifuging to obtain an enzymolysis solution;
(3) degrading polysaccharide: adding an auxiliary degrading agent into the enzymolysis liquid obtained in the step (2) and carrying out ultrasonic treatment to obtain degradation liquid;
(4) ultrafiltration and desugarization: and (4) selecting an ultrafiltration membrane with the molecular weight of 5-10kDa to filter the degradation liquid obtained in the step (3), and collecting filtrate to obtain a type II collagen peptide solution.
As a further preference of the present invention, the volume ratio of the auxiliary degradation agent to the enzymolysis liquid in the step (3) is 1-2.5: 25; the auxiliary degradation agent is one or a compound of hydrogen peroxide and ethanol.
As a further preferred aspect of the present invention, the ultrasonic treatment conditions in step (3) are: the ultrasonic frequency is 20-80kHz, the temperature is 30-60 ℃, and the time is 0.5-3 h.
As a further preference of the present invention, the filtrate flow rate in step (4) is 2 to 4 ml/min.
As a further preferred aspect of the present invention, the mass ratio of the protease to the animal cartilage in the step (2) is 0.01 to 0.1: 1.
as a further preferred aspect of the present invention, the protease in step (2) includes alkaline protease, neutral protease, and pepsin.
As a further preferred aspect of the present invention, the degreasing treatment in the step (1) comprises: soaking in ether solution for 12-24 hr, and heating until ether is completely volatilized.
As a further preferred aspect of the present invention, the dehybridization treatment in step (1) comprises: adding NaOH solution at 0-10 deg.C, soaking and magnetically stirring for 3-12h, and replacing NaOH solution every 2-3 h.
As a further preferable mode of the present invention, the decalcification treatment in step (1) is performed using an EDTA decalcification solution, wherein the volume ratio of the EDTA decalcification solution to the animal cartilage is 4-10: 1.
In conclusion, the invention has the following beneficial effects:
the method for preparing the II type collagen peptide by adopting the process of combining the hydrogen peroxide ultrasonic degradation and the ultrafiltration membrane technology has the advantages of high desugarization efficiency, no organic solvent residue and low protein peptide loss rate, improves the processing utilization rate of the animal cartilage, is green and pollution-free in the whole processing process, has the characteristics of good economic benefit and environmental friendliness, and has good application and development prospects.
Detailed Description
A method for removing type II collagen peptide polysaccharide comprises the following steps:
(1) pretreatment of raw materials: selecting animal cartilage from which chondroitin sulfate is extracted, crushing, and then carrying out degreasing, impurity protein removal and decalcification treatment.
The animal cartilage from which the chondroitin sulfate is extracted is a raw material of the method, the process for extracting the chondroitin sulfate from the animal cartilage is the prior art in the field, and the specific steps are not limited (refer to the process flow for extracting the chondroitin sulfate disclosed in the patent application No. 201911423904.4).
The degreasing treatment adopts ether solution, because ether has the characteristics of dissolving fat and quickly volatilizing at the temperature of about 40 ℃, the degreasing purpose can be achieved, reagent residue cannot be caused, and the activity of the protein peptide with high heat sensitivity cannot be influenced by the condition of low-temperature volatilization, so that the ether solution is preferred.
The above-mentioned dehybridization treatment comprises: adding NaOH solution at 0-10 deg.C, soaking and magnetically stirring for 3-12h, and replacing NaOH solution every 2-3 h. The principle that the method can effectively remove the foreign protein is that the pH value of the solution is controlled by the NaOH solution, so that the foreign protein on the cartilage surface is dissolved in the NaOH solution and is completely removed along with the replacement of the NaOH solution. The purpose of magnetic stirring is to accelerate the dissolution of the hybrid protein, the rotating speed is controlled within the range of 100-500r/min, and the hybrid protein precipitate generated by high-speed rotation is avoided from being difficult to remove.
The above-mentioned decalcification treatment adopts EDTA decalcification solution, the volume ratio of said EDTA decalcification solution and animal cartilage is 4-10: 1; the preparation of EDTA decalcification solution belongs to the prior art, and is not described in detail.
(2) Preparing enzymolysis liquid: adding protease into the pretreated animal cartilage for enzymolysis for 2-6h, inactivating enzyme, and centrifuging to obtain enzymatic hydrolysate; wherein the protease is selected from alkaline protease, neutral protease, and pepsin, or other commonly used protease such as papain, trypsin, etc.
(3) Degrading polysaccharide: adding an auxiliary degrading agent into the enzymolysis liquid and carrying out ultrasonic treatment to obtain degradation liquid; the auxiliary degradation agent can be selected from one or a combination of hydrogen peroxide and ethanol, and is preferably hydrogen peroxide. The method adopts the technology of degrading the polysaccharide by hydrogen peroxide ultrasonic, so that the molecular weight of the polysaccharide can be effectively reduced, an ultrafiltration membrane with smaller pore diameter can be used for one-step removal, the problem of ultrafiltration membrane blockage can not occur, the removal efficiency can be improved, and the loss rate of protein peptide can be reduced.
(4) Ultrafiltration and desugarization: and (3) selecting an ultrafiltration membrane with the molecular weight of 3-10kDa to filter the degradation liquid, and collecting filtrate to obtain the II type collagen peptide solution. The ultrafiltration membrane in the process needs to be treated, and comprises the following steps: soaking the ultrafiltration membrane in pure water for 4-6h, changing water for three times, and washing the ultrafiltration membrane with pure water to neutrality for use. And a pipeline valve used by the inspection equipment ensures smooth operation of the pipeline.
Example 1
(1) Pretreatment of cartilage: unfreezing the animal cartilage from which the chondroitin sulfate is extracted, crushing the animal cartilage by a crusher, cleaning the animal cartilage by tap water, and then carrying out degreasing, impurity protein removal and decalcification treatment according to the following steps. A. Adding ether solution with volume 10 times of that of cartilage, soaking for 24h for degreasing, and completely volatilizing ether on the surface of cartilage at 40 ℃; B. adding 20 times of NaOH solution with the concentration of 0.25mol/L at the temperature of 4 ℃, stirring for 12 hours on a magnetic stirrer, and changing the solution about every 3 hours; C. the cartilage was decalcified by adding a 0.5mol/L EDTA solution in a volume 10 times the volume of the cartilage.
(2) Obtaining an enzymolysis liquid: adding 4% of pepsin into the pretreated cartilage, dissolving the pepsin according to the material-liquid ratio of 1:8, adjusting the pH to be the most suitable, carrying out enzymolysis for 3.5 hours at the constant temperature of 40 ℃, and inactivating the enzyme for 10 minutes in a boiling water bath after the enzymolysis is finished; and after the enzymolysis liquid is cooled, adjusting the pH value of the enzymolysis liquid to be neutral, and performing centrifugal separation to obtain the animal cartilage enzymolysis liquid.
(3) Degrading polysaccharide: adding an ultrasonic auxiliary degradation agent into the enzymatic hydrolysate to enable the volume fraction to reach 4%, putting the enzymatic hydrolysate into an ultrasonic device, and adjusting the ultrasonic conditions as follows: the power is 30kHz, the reaction temperature is 40 ℃, and the reaction time is 1 h.
(4) And (3) desugarizing: selecting an ultrafiltration membrane with a molecular weight of 5kDa, soaking the ultrafiltration membrane in pure water for 4-6h, changing water for three times in the middle, and washing the ultrafiltration membrane to be neutral by using the pure water for later use. And a pipeline valve used by the inspection equipment ensures smooth operation of the pipeline. Directly connecting a hose at an air inlet at the top end of the ultrafiltration cup with a nitrogen pipeline in a fume hood, placing the ultrafiltration cup on a magnetic stirrer, opening the magnetic stirrer to enable a stirrer to rotate at a proper rotating speed, adding enzymatic hydrolysate from a liquid inlet at the top end of the ultrafiltration cup, and screwing a screw cap to keep the airtightness of the ultrafiltration cup. Opening the piston at the top of the nitrogen cylinder, opening the stop valve of the nitrogen pipeline to the maximum, adjusting the switch leading to the fume hood to ensure that the nitrogen pressure is 0.35-0.5MPa, rotating clockwise to slowly adjust the nitrogen pressure to ensure that the flow rate of filtrate is 3-4ml/min, and collecting filtrate. After use, the ultrafiltration membrane is soaked in 0.5mol/L NaOH for 10-15min, and then is taken out and washed to be neutral by pure water.
Example 2
(1) Pretreatment of cartilage: unfreezing the animal cartilage from which the chondroitin sulfate is extracted, crushing the animal cartilage by a crusher, cleaning the animal cartilage by tap water, and then carrying out degreasing, impurity protein removal and decalcification treatment according to the following steps. A. Adding ether solution with volume 8 times of cartilage volume, soaking for 18h for defatting, and completely volatilizing ether on cartilage surface at 40 deg.C; B. adding 16 times of NaOH solution with the concentration of 0.25mol/L at the temperature of 4 ℃, stirring for 9 hours on a magnetic stirrer, and changing the solution about every 3 hours; C. the cartilage was decalcified by adding 0.5mol/L EDTA solution in 4 times the volume of the cartilage.
(2) Obtaining an enzymolysis liquid: adding 6% of neutral protease into the pretreated cartilage, dissolving according to a material-liquid ratio of 1:10, adjusting to the most suitable pH, performing enzymolysis at a constant temperature of 45 ℃ for 3h, and inactivating the enzyme in a boiling water bath for 10 min after the enzymolysis is finished; and after the enzymolysis liquid is cooled, adjusting the pH value of the enzymolysis liquid to be neutral, and performing centrifugal separation to obtain the animal cartilage enzymolysis liquid.
(3) Degrading polysaccharide: adding an ultrasonic auxiliary degradation agent into the enzymatic hydrolysate to enable the volume fraction to reach 10%, putting the enzymatic hydrolysate into an ultrasonic device, and adjusting the ultrasonic conditions as follows: the power is 40kHz, the reaction temperature is 55 ℃, and the reaction time is 2 h.
(4) And (3) desugarizing: selecting an ultrafiltration membrane with a molecular weight of 10kDa, soaking the ultrafiltration membrane in pure water for 4-6h, changing water for three times in the middle, and washing the ultrafiltration membrane to be neutral by using the pure water for later use. And a pipeline valve used by the inspection equipment ensures smooth operation of the pipeline. Directly connecting a hose at an air inlet at the top end of the ultrafiltration cup with a nitrogen pipeline in a fume hood, placing the ultrafiltration cup on a magnetic stirrer, opening the magnetic stirrer to enable a stirrer to rotate at a proper rotating speed, adding enzymatic hydrolysate from a liquid inlet at the top end of the ultrafiltration cup, and screwing a screw cap to keep the airtightness of the ultrafiltration cup. Opening the piston at the top of the nitrogen cylinder, opening the stop valve of the nitrogen pipeline to the maximum, adjusting the switch leading to the fume hood to ensure that the nitrogen pressure is 0.2-0.3MPa, rotating clockwise to slowly adjust the nitrogen pressure to ensure that the flow rate of filtrate is 2-3ml/min, and collecting filtrate. After use, the ultrafiltration membrane is soaked in 0.5mol/L NaOH for 10-15min, and then is taken out and washed to be neutral by pure water.
Comparative example 1
(1) Pretreatment of cartilage: unfreezing the animal cartilage from which the chondroitin sulfate is extracted, crushing the animal cartilage by a crusher, cleaning the animal cartilage by tap water, and then carrying out degreasing, impurity protein removal and decalcification treatment according to the following steps. A. Adding ether solution with volume 10 times of cartilage volume, soaking for 12h for defatting, and completely volatilizing ether on cartilage surface at 40 deg.C; B. adding 12 times of NaOH solution with the concentration of 0.25mol/L at the temperature of 4 ℃, stirring for 12 hours on a magnetic stirrer, and changing the solution about every 3 hours; C. the cartilage was decalcified by adding a 6-fold volume of 0.5mol/L EDTA solution.
(2) Obtaining an enzymolysis liquid: adding 2% of alkaline protease into the pretreated cartilage, dissolving according to a material-liquid ratio of 1:4, adjusting to the most suitable pH, performing enzymolysis at a constant temperature of 35 ℃ for 5h, and inactivating the enzyme in a boiling water bath for 10 min after the enzymolysis is finished; and after the enzymolysis liquid is cooled, adjusting the pH value of the enzymolysis liquid to be neutral, and performing centrifugal separation to obtain the animal cartilage enzymolysis liquid.
(3) And (3) desugarizing: respectively sucking 5mL of the enzymolysis solution and 30mL of 50% alcohol solution, mixing uniformly, sticking a sealing film, and standing in a refrigerator at 4 ℃ for 24 h.
Comparative example 2
(1) Pretreatment of cartilage: unfreezing the animal cartilage from which the chondroitin sulfate is extracted, crushing the animal cartilage by a crusher, cleaning the animal cartilage by tap water, and then carrying out degreasing, impurity protein removal and decalcification treatment according to the following steps. A. Adding ether solution 6 times the volume of cartilage, soaking for 20h for defatting, and completely volatilizing ether at 40 deg.C; B. adding 10 times of NaOH solution with the concentration of 0.25mol/L at the temperature of 4 ℃, stirring for 12 hours on a magnetic stirrer, and changing the solution about every 3 hours; C. the cartilage was decalcified by adding a 0.5mol/L EDTA solution in a volume 10 times the volume of the cartilage.
(2) Obtaining an enzymolysis liquid: adding 4% of alkaline protease into the pretreated cartilage, dissolving according to a material-liquid ratio of 1:6, adjusting to the optimum pH, performing enzymolysis at a constant temperature of 40 ℃ for 4h, heating to 90 ℃ after the enzymolysis is finished, and inactivating the enzyme in a water bath for 15 min; and after the enzymolysis liquid is cooled, adjusting the pH value of the enzymolysis liquid to be neutral, and performing centrifugal separation to obtain the animal cartilage enzymolysis liquid.
(3) And (3) desugarizing: respectively sucking 10mL of the enzymolysis liquid and 60mL of 70% alcohol solution, mixing uniformly, sticking a sealing film, and standing in a refrigerator at 4 ℃ for 24 h.
Comparative example 3:
(1) pretreatment of cartilage: unfreezing the animal cartilage after extracting the chondroitin sulfate, crushing the animal cartilage by a crusher, cleaning the animal cartilage by tap water, and carrying out degreasing, impurity protein removal and decalcification treatment according to the following steps. A. Adding ether solution with volume 10 times of that of cartilage, soaking for 24h for degreasing, and completely volatilizing ether on the surface of cartilage at 40 ℃; B. adding 12 times of NaOH solution with the concentration of 0.25mol/L at the temperature of 4 ℃, stirring for 6 hours on a magnetic stirrer, and changing the solution about every 3 hours; C. the cartilage was decalcified by adding 0.5mol/L EDTA solution 8 times the volume of the cartilage.
(2) Obtaining an enzymolysis liquid: adding 6% of alkaline protease into the pretreated cartilage, dissolving according to a material-liquid ratio of 1:8, adjusting to the optimum pH, performing enzymolysis at a constant temperature of 45 ℃ for 3h, heating to 90 ℃ after the enzymolysis is finished, and inactivating the enzyme in a water bath for 20 min; and after the enzymolysis liquid is cooled, adjusting the pH value of the enzymolysis liquid to be neutral, and performing centrifugal separation to obtain the animal cartilage enzymolysis liquid.
(3) And (3) desugarizing: respectively sucking 15 mL of the enzymolysis solution and 90mL of 80% alcohol solution, mixing uniformly, sticking a sealing film, and standing in a refrigerator at 4 ℃ for 24 h.
Comparative example 4
This comparative example differs from example 1 in that a 3kDa molecular weight ultrafiltration membrane was chosen.
The results of examining the desugarization rate (%) and the polypeptide loss rate (%) of the enzymatic hydrolysates obtained in examples 1 to 2 and comparative examples 1 to 4 were as follows:
as shown above, comparative examples 1 to 3 show that with the increase of the desugarization rate, the loss rate of the polypeptide is also greatly increased, and the effects of high polysaccharide removal rate and low polypeptide loss rate cannot be achieved. The embodiment 1-2 adopting the process of the invention can simultaneously achieve the effects of high polysaccharide removal rate and low polypeptide loss rate. Comparative example 4 no expected effect was achieved with the 3kda molecular weight ultrafiltration membrane, probably because the choice of pore size for the ultrafiltration membrane had a greater effect on the rate of polypeptide loss.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (9)
1. A method for removing type II collagen peptide polysaccharide, which is characterized by comprising the following steps:
(1) pretreatment of raw materials: selecting animal cartilage from which chondroitin sulfate is extracted, crushing, and then carrying out degreasing, impurity protein removal and decalcification treatment;
(2) preparing enzymolysis liquid: adding protease into the animal cartilage obtained by the pretreatment in the step (1) for enzymolysis for 2-6h, and then inactivating the enzyme and centrifuging to obtain enzymatic hydrolysate;
(3) degrading polysaccharide: adding an auxiliary degrading agent into the enzymolysis liquid obtained in the step (2) and carrying out ultrasonic treatment to obtain degradation liquid;
(4) ultrafiltration and desugarization: and (4) selecting an ultrafiltration membrane with the molecular weight of 5-10kDa to filter the degradation liquid obtained in the step (3), and collecting filtrate to obtain a type II collagen peptide solution.
2. The method for removing type II collagen peptidoglycan according to claim 1, wherein the volume ratio of the auxiliary degradation agent to the enzymolysis solution in step (3) is 1-2.5: 25; the auxiliary degradation agent is one or a compound of hydrogen peroxide and ethanol.
3. The method for removing type II collagen peptidoglycan according to claim 2, wherein the ultrasonication treatment in step (3) is performed under the following conditions: the ultrasonic frequency is 20-80kHz, the temperature is 30-60 ℃, and the time is 0.5-3 h.
4. The method for removing type II collagen peptidoglycan according to claim 1, wherein the flow rate of the filtrate in step (4) is 2-4 ml/min.
5. The method for removing type II collagen peptidoglycan according to claim 1, wherein the mass ratio of the protease to the animal cartilage in step (2) is 0.01-0.1: 1.
6. the method for removing type II collagen peptidoglycan according to claim 5, wherein the protease in step (2) comprises alkaline protease, neutral protease, or pepsin.
7. The method of claim 1, wherein the step (1) of degreasing comprises: soaking in diethyl ether solution for 12-24 hr, and heating until diethyl ether is completely volatilized.
8. The method of claim 1, wherein the dehiscent treatment of collagen type II peptidoglycan in step (1) comprises: adding NaOH solution at 0-10 deg.C, soaking and magnetically stirring for 3-12h, and replacing NaOH solution every 2-3 h.
9. The method for removing type II collagen peptidoglycan according to claim 1, wherein the decalcification treatment in step (1) is performed using EDTA decalcification solution, and the volume ratio of EDTA decalcification solution to animal cartilage is 4-10: 1.
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