CN105331662A - Non-denatured II type collagen of animal cartilage source and preparation method for non-denatured II type collagen - Google Patents
Non-denatured II type collagen of animal cartilage source and preparation method for non-denatured II type collagen Download PDFInfo
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Abstract
The invention discloses a preparation method for non-denatured II type collagen of an animal cartilage source. The preparation method is characterized by comprising the following steps: taking fresh and traceable animal cartilages as raw materials; carrying out purification treatment on the animal cartilages by virtue of processes of repeated freeze-thawing, ultrasonic degreasing, hypertonic solution decellularizing, acid-base softening and the like; carrying out processes of guanidine hydrochloride sugar-removing, pickling, compound enzyme treatment, multi-time salting-out, multi-time centrifugal purification, membrane dialysis, freeze-drying and the like, thereby obtaining spongy non-denatured II type collagen. The high-purity high-yield II type collagen obtained by the preparation method keeps the original complete triple-helix structure of the collagen, is high in biological activity, stable in structure, easy to store and beneficial to adhesion, growth and multiplication of cartilage cells, and can be used for preparing medical biological materials.
Description
Technical field
The present invention relates to a kind of preparation method of non-denatured type II collagen of animal cartilage source, belong to bio-medical material preparation field.
Background technology
II Collagen Type VI is one of main component forming cartilage matrix, it is similar to type i collagen, processbearing astrocyte collagen, form triple helix structural domain by three identical α I (II) chains, its molecule is not containing tryptophan residue, and tyrosine residues content is also less, therefore the antigenicity of its molecule is lower, especially the II Collagen Type VI that enzyme process is obtained, after eliminating the end peptide of molecular chain, its immunogenicity is lower.Put up a bridge by covalent linkage between II Collagen Type VI molecule in cartilage crosslinked; form stable tridimensional network; the hydroxylysine that II Collagen Type VI is rich in closes with polysaccharide covalent bond again; more stabilize the natural space structure of II Collagen Type VI; this is conducive to the protection structure of cartilage and the biological activity of cartilage, and maintains mechanical property and the mechanical stability of natural cartilage.Meanwhile, it is very low that the space structure of natural II Collagen Type VI determines its solubleness in water, is unfavorable for the separating-purifying of II Collagen Type VI.Therefore in leaching process, maintain the triple helix structure of collagen well, not reason excess processes and collagen is decomposed further and loses possessed physiologically active, seem particularly important.Up to now, the extraction preparation method of II Collagen Type VI is mainly acid system, alkaline process, enzyme process, combined techniques, neutral sulfity process and hot-water extraction method etc. six kinds, the molecular structure of the II Collagen Type VI obtained by often kind of method and performance exist significantly to be distinguished, wherein, alkaline process, hot-water extraction method gained II Collagen Type VI molecular weight are low, wider distribution, do not possess biological activity; Acid system, neutral sulfity process gained II Collagen Type VI, because not operatively removing its molecular end peptide, II Collagen Type VI may be made to have certain immunogenicity, affect its biological applications, and gained collagen yield are too low, not easily suitability for industrialized production; Its molecular structure of the complete reservation of II Collagen Type VI energy of Enzymatic, extraction yield is also relatively high, the most applicable scale operation.But there is contradiction between the purity of enzyme process collagen in preparation II Collagen Type VI process and productive rate, if productive rate is high, purity will not reach requirement, therefore takes into account high yield and highly purified II Collagen Type VI extracting and preparing technique is most important.
Summary of the invention
The preparation method of the non-denatured type II collagen in a kind of animal cartilage source provided for the deficiencies in the prior art is provided, be characterized in that unmodified II collagen productive rate prepared by the method, purity are high, biological activity is high, Stability Analysis of Structures is easy to preserve, be conducive to the sticking of chondrocyte, grow, breed, can be used for the preparation of biomaterial for medical purpose.
For achieving the above object, the present invention adopts following technical scheme:
(1) pre-treatment of animal cartilage: get the fresh animal cartilage of tracing to the source 100 ~ 200 parts, remain bone, muscle with scissors manual removal, then repeatedly clean 3 ~ 5 times with physiological saline in stainless steel rotary drum, draining 30 ~ 60min; Animal cartilage is put into the quick-freeze refrigerator multigelation 5 ~ 10 times of-40 ~-80 DEG C, then be immersed in the grease-removing agent of 1000 ~ 10000 parts by volume, adjoint intermittent power is 50 ~ 100W ultrasonic wave, often act on 30 ~ 60min and stop 10-30min, repeated action 3 ~ 5 times, liquid is changed 2 ~ 5 times in midway, with 0.05MTris, 1MNaCl, pH is the Tris-NaCl buffered soln cleaning 3 ~ 5 times of 7.5, then is immersed in 2 ~ 3.5MNaCl, in the hypertonic solution of 20 ~ 40mMEDTA, 10 ~ 20h, draining 30 ~ 60min is stirred at 37 DEG C; Animal cartilage is continued to be immersed in pH2.0 ~ 2.5, acetum 2 ~ the 10h of 1000 ~ 5000 parts by volume, filtered through gauze recovery of acetic acid solution, washed with de-ionized water 3 ~ 5 times, with 0.1 ~ 1M, the NaOH solution sofening treatment 10-24h of 1000 ~ 5000 parts by volume, washed with de-ionized water 3 ~ 5 times, lyophilize, obtains the animal cartilage of purifying; Adopted by animal cartilage superfine comminution at low temperature machine to pulverize, preserve stand-by in moisture eliminator;
(2) extraction of non-denatured type II collagen: 100 ~ 200 parts of animal cartilage powder are immersed in the 0.05MTris that 1000 ~ 2000 parts by volume contain 4M Guanidinium hydrochloride, 1MNaCl, pH is in the Tris-NaCl buffered soln of 7.5, stirred at ambient temperature 10 ~ 36h, then the centrifugal 10 ~ 30min of 10000 ~ 20000rpm, reclaim supernatant liquor, with deionized water washing and precipitating thing repeatedly, throw out is immersed in pH2.0 ~ 2.5, the acetum of 3000 ~ 10000 parts by volume, slowly 3 ~ 10h is stirred at constant temperature 4 ~ 10 DEG C, then 1 ~ 4 part of prozyme is added, 24 ~ 72h is rotated at 4 ~ 10 DEG C, obtain enzyme molten animal cartilage II Collagen Type VI solution,
(3) purifying of non-denatured type II collagen: to above-mentioned gained to animal cartilage II Collagen Type VI solution in add the sodium chloride powder that ultimate density is 0.9mol/L, 10 ~ 16h is stirred at 4 ~ 10 DEG C, centrifugal 10 ~ the 30min of 10000 ~ 20000rpm, abandon supernatant liquid, throw out is dissolved in 0.1 ~ 0.5mol/L acetum again, be 6.5 ~ 7.5 by 5 ~ 10MNaOH solution adjust ph, add the sodium chloride powder that ultimate density is 4mol/L, 10 ~ 16h is stirred at 4 ~ 10 DEG C, then the centrifugal 10 ~ 30min of 10000 ~ 20000rpm, abandon supernatant liquid, throw out is dissolved in 0.1 ~ 0.5mol/L acetum again, be 6.5 ~ 7.5 by 5 ~ 10MNaOH solution adjust ph again, add the sodium chloride powder that ultimate density is 4mol/L, 10 ~ 16h is stirred at 4 ~ 10 DEG C, again with the centrifugal 10 ~ 30min of 10000 ~ 20000rpm, take off a layer throw out, so repeatedly saltout 3 ~ 5 times, the precipitation obtained after saltouing is dissolved in 0.1 ~ 0.5mol/L acetum again, take concentration as the acetic acid solution of 0.1 ~ 0.5M is dialyzate dialysis treatment 2-3 days, and freeze-drying, obtains spongy unmodified animal cartilage II Collagen Type VI, obtained animal cartilage II Collagen Type VI, its key property reaches following index request:
Outward appearance: white sponge, the impurity visible without naked eyes and variable color;
Tryptophan analysis: should not tryptophane be contained;
Heavy metal content :≤10 μ g/g (m/m);
Hydroxyproline content: 8.5% (m/m) that should be not less than total protein content;
Liposome :≤1% (m/m);
Ash content :≤2% (m/m);
Foreign protein content :≤1% (m/m);
Cytotoxicity: cell-cytotoxic reaction is not more than 1 grade;
Sterility test: aseptic;
Sensitization test: without delayed hypersensitivity;
Intradermoreaction is tested: primary stimulation index PII<0.4.
In above-mentioned preparation method, superfine comminution at low temperature machine in step (1), Hao Yu development in science and technology company limited of BeiJing ZhongKe produces; Ultrasonic time looks the difference of ultrasonic power used and different in step (1); In step (1), in prozyme, pepsic unit of activity is 3000 units/gram, and Papain Enzymatic Activity unit is 8,000,000 units/gram, and the unit of activity of dextranase is 50 units/gram; Preparation process all completes at 4 ~ 10 DEG C.
The art of this patent is taken off the techniques such as cell, acid-alkali alternately soften and is carried out pre-treatment by multigelation, ultrasonic degreasing, high sepage to animal cartilage, effectively can remove the non-collagen tissue of cartilage, collegen filament in loose further animal cartilage, space between the collegen filament adding animal cartilage, be highly advantageous to the abundant infiltration of biological enzyme in leaching process and effect, effectively can improve purity and the productive rate of collagen; The synergy of each component of prozyme, can remove the end peptide of collagen to greatest extent, reduce its immunogenicity.
non-denatured type II collagen has following purposes:
1. be used as the raw material of biomedical material, prepare the raw material of medicine controlled release carrier, hemostatic material, endocranium repair materials, tissue engineering bracket, tissue guiding materials, shaping and beauty material etc. as can be used as;
2. be used as the raw material of cosmetics material, as the raw material of protective skin cream, a profit agent etc.;
3. be used as the raw material of foodstuffs industry material, as the raw material of health-care material, beverage etc.;
4. other, as be used as cell cultures use, bio-reactor monomer film etc.
compared with prior art, tool has the following advantages this technology:
(1) the present invention employs ultrasonic wave in animal cartilage purge process, has given full play to the effects such as hyperacoustic cavitation effect, mechanical effect, heat effect, has destroyed the cytolemma of cell in cartilage, be conducive to the further degreasing to cartilage and Cell extraction;
(2) the present invention adopts multiplex-enzyme extraction II Collagen Type VI, and the synergy between prozyme component is highly advantageous to and removes the non-collagen tissue in animal cartilage, heteroproteose cell and collagen end peptide, effectively can improve productive rate and the purity of collagen;
(3) II Collagen Type VI preparation technology of the present invention, first animal cartilage is taken off the techniques such as cell, acid-alkali alternately soften carried out purification process by multigelation, ultrasonic degreasing, high sepage, fully eliminate the non-collagen tissue in cartilage, collegen filament in loose cartilage, can improve productive rate and the purity of collagen effectively;
(4) drawing materials of the II Collagen Type VI prepared by the present invention is abundant, with low cost, and advanced technology is reliable, and output is large, is applicable to batch production.
Embodiment
Below by enforcement, the present invention is specifically described; what be necessary to herein means out is that the present embodiment is only used to further illustrate the present invention; and limiting the scope of the invention can not be interpreted as, the person skilled in the art in this field can make nonessential improvement and adjustment according to the content of foregoing invention.
Embodiment 1
(1) pre-treatment of animal cartilage: get the fresh animal cartilage of tracing to the source 100 parts, remain bone, muscle with scissors manual removal, then repeatedly clean 3 times with physiological saline, draining 30min; Animal cartilage is put into the quick-freeze refrigerator multigelation 5 times of-40 DEG C, then being immersed in stainless steel rotary drum in the grease-removing agent of 1000 parts by volume, is that 50W ultrasonic wave often acts on 60min and stops 30min, repeated action 5 times with intermittent power, liquid is changed 5 times in midway, 3 times are cleaned with the Tris-NaCl buffered soln that 0.05MTris, 1MNaCl, pH are 7.5, be immersed in 2MNaCl again, in the hypertonic solution of 20mMEDTA, at 37 DEG C, stir 10h, draining 30min; Animal cartilage is continued to be immersed in pH2.0, the acetum 2h of 1000 parts by volume, filtered through gauze recovery of acetic acid solution, washed with de-ionized water 3 times, with 0.1M, the NaOH solution sofening treatment 10h of 1000 parts by volume, washed with de-ionized water 3 times, lyophilize, obtains the animal cartilage of purifying; Adopted by animal cartilage superfine comminution at low temperature machine to pulverize, preserve stand-by in moisture eliminator;
(2) extraction of non-denatured type II collagen: 100 parts of animal cartilage powder are immersed in the 0.05MTris that 1000 parts by volume contain 4M Guanidinium hydrochloride, 1MNaCl, pH is in the Tris-NaCl buffered soln of 7.5, stirred at ambient temperature 10h, then the centrifugal 30min of 10000rpm, reclaim supernatant liquor, with deionized water washing and precipitating thing repeatedly, throw out is immersed in pH2.0, the acetum of 3000 parts by volume, slowly stirs 3h at constant temperature 4 DEG C, then adds 1 part of prozyme, rotate 72h at 4 DEG C, obtain enzyme molten animal cartilage II Collagen Type VI solution;
(3) purifying of non-denatured type II collagen: to above-mentioned gained to animal cartilage II Collagen Type VI solution in add the sodium chloride powder that ultimate density is 0.9mol/L, 10h is stirred at 4 DEG C, the centrifugal 30min of 10000rpm, abandon supernatant liquid, throw out is dissolved in 0.1mol/L acetum again, be 6.5 by 5MNaOH solution adjust ph, add the sodium chloride powder that ultimate density is 4mol/L, 10h is stirred at 4 DEG C, then the centrifugal 30min of 10000rpm, abandon supernatant liquid, throw out is dissolved in 0.1mol/L acetum again, be 6.5 by 5MNaOH solution adjust ph again, add the sodium chloride powder that ultimate density is 4mol/L, 10h is stirred at 4 DEG C, again with the centrifugal 30min of 10000rpm, take off a layer throw out, so repeatedly saltout 3 times, the precipitation obtained after saltouing is dissolved in 0.1mol/L acetum again, take concentration as the acetic acid solution of 0.1M is dialyzate dialysis treatment 2 days, and freeze-drying, obtains spongy unmodified animal cartilage II Collagen Type VI.
Embodiment 2
(1) pre-treatment of animal cartilage: get the fresh animal cartilage of tracing to the source 150 parts, remain bone, muscle with scissors manual removal, then repeatedly clean 4 times with physiological saline in stainless steel rotary drum, draining 40min; Animal cartilage is put into the quick-freeze refrigerator multigelation 8 times of-50 DEG C, then being immersed in the grease-removing agent of 4500 parts by volume, is that 60W ultrasonic wave often acts on 40min and stops 20min, repeated action 4 times with intermittent power, liquid is changed 3 times in midway, 4 times are cleaned with the Tris-NaCl buffered soln that 0.05MTris, 1MNaCl, pH are 7.5, be immersed in 3MNaCl again, in the hypertonic solution of 30mMEDTA, at 37 DEG C, stir 15h, draining 40min; Animal cartilage is continued to be immersed in pH2.2, the acetum 8h of 4500 parts by volume, filtered through gauze recovery of acetic acid solution, washed with de-ionized water 4 times, with 0.5M, the NaOH solution sofening treatment 18h of 4500 parts by volume, washed with de-ionized water 4 times, lyophilize, obtains the animal cartilage of purifying; Adopted by animal cartilage superfine comminution at low temperature machine to pulverize, preserve stand-by in moisture eliminator;
(2) extraction of non-denatured type II collagen: 150 parts of animal cartilage powder are immersed in the 0.05MTris that 1500 parts by volume contain 4M Guanidinium hydrochloride, 1MNaCl, pH is in the Tris-NaCl buffered soln of 7.5, stirred at ambient temperature 10 ~ 36h, then the centrifugal 20min of 15000rpm, reclaim supernatant liquor, with deionized water washing and precipitating thing repeatedly, throw out is immersed in pH2.2, the acetum of 4500 parts by volume, slowly stirs 5h at constant temperature 5 DEG C, then adds 3 parts of prozymes, rotate 36h at 5 DEG C, obtain enzyme molten animal cartilage II Collagen Type VI solution;
(3) purifying of non-denatured type II collagen: to above-mentioned gained to animal cartilage II Collagen Type VI solution in add the sodium chloride powder that ultimate density is 0.9mol/L, 14h is stirred at 5 DEG C, the centrifugal 20min of 15000rpm, abandon supernatant liquid, throw out is dissolved in 0.2mol/L acetum again, be 7.0 by 8MNaOH solution adjust ph, add the sodium chloride powder that ultimate density is 4mol/L, 12h is stirred at 5 DEG C, then the centrifugal 20min of 15000rpm, abandon supernatant liquid, throw out is dissolved in 0.2mol/L acetum again, be 7.0 by 8MNaOH solution adjust ph again, add the sodium chloride powder that ultimate density is 4mol/L, 12h is stirred at 5 DEG C, again with the centrifugal 20min of 15000rpm, take off a layer throw out, so repeatedly saltout 4 times, the precipitation obtained after saltouing is dissolved in 0.2mol/L acetum again, take concentration as the acetic acid solution of 0.2M is dialyzate dialysis treatment 2 days, and freeze-drying, obtains spongy unmodified animal cartilage II Collagen Type VI.
Embodiment 3
(1) pre-treatment of animal cartilage: get the fresh animal cartilage of tracing to the source 200 parts, remain bone, muscle with scissors manual removal, then repeatedly clean 5 times with physiological saline, draining 60min; Animal cartilage is put into the quick-freeze refrigerator multigelation 10 times of-80 DEG C, then being immersed in stainless steel rotary drum in the grease-removing agent of 10000 parts by volume, is that 100W ultrasonic wave often acts on 30min and stops 10min, repeated action 3 times with intermittent power, liquid is changed 2 times in midway, 5 times are cleaned with the Tris-NaCl buffered soln that 0.05MTris, 1MNaCl, pH are 7.5, be immersed in 3.5MNaCl again, in the hypertonic solution of 40mMEDTA, at 37 DEG C, stir 20h, draining 60min; Animal cartilage is continued to be immersed in pH2.5, the acetum 2h of 5000 parts by volume, filtered through gauze recovery of acetic acid solution, washed with de-ionized water 5 times, with 1M, the NaOH solution sofening treatment 24h of 5000 parts by volume, washed with de-ionized water 5 times, lyophilize, obtains the animal cartilage of purifying; Adopted by animal cartilage superfine comminution at low temperature machine to pulverize, preserve stand-by in moisture eliminator;
(2) extraction of non-denatured type II collagen: 200 parts of animal cartilage powder are immersed in the 0.05MTris that 2000 parts by volume contain 4M Guanidinium hydrochloride, 1MNaCl, pH is in the Tris-NaCl buffered soln of 7.5, stirred at ambient temperature 36h, then the centrifugal 10min of 20000rpm, reclaim supernatant liquor, with deionized water washing and precipitating thing repeatedly, throw out is immersed in pH2.5, the acetum of 10000 parts by volume, slowly stirs 10h at constant temperature 10 DEG C, then adds 4 parts of prozymes, rotate 24h at 10 DEG C, obtain enzyme molten animal cartilage II Collagen Type VI solution;
(3) purifying of non-denatured type II collagen: to above-mentioned gained to animal cartilage II Collagen Type VI solution in add the sodium chloride powder that ultimate density is 0.9mol/L, 16h is stirred at 10 DEG C, the centrifugal 10min of 20000rpm, abandon supernatant liquid, throw out is dissolved in 0.5mol/L acetum again, be 7.5 by 10MNaOH solution adjust ph, add the sodium chloride powder that ultimate density is 4mol/L, 16h is stirred at 10 DEG C, then the centrifugal 10min of 20000rpm, abandon supernatant liquid, throw out is dissolved in 0.5mol/L acetum again, be 7.5 by 10MNaOH solution adjust ph again, add the sodium chloride powder that ultimate density is 4mol/L, 16h is stirred at 10 DEG C, again with the centrifugal 10min of 20000rpm, take off a layer throw out, so repeatedly saltout 5 times, the throw out obtained after saltouing is dissolved in 0.5mol/L acetum again, take concentration as the acetic acid solution of 0.5M is dialyzate dialysis treatment 3 days, and freeze-drying, obtains spongy unmodified animal cartilage II Collagen Type VI.
Claims (4)
1. a preparation method for the non-denatured type II collagen in animal cartilage source, is characterized in that the method comprises the following steps: described raw material number by weight
(1) pre-treatment of animal cartilage: get the fresh animal cartilage of tracing to the source 100 ~ 200 parts, remain bone, muscle with scissors manual removal, then repeatedly clean 3 ~ 5 times with physiological saline in stainless steel rotary drum, draining 30 ~ 60min; Animal cartilage is put into the quick-freeze refrigerator multigelation 5 ~ 10 times of-40 ~-80 DEG C, then be immersed in the grease-removing agent of 1000 ~ 10000 parts by volume, adjoint intermittent power is 50 ~ 100W ultrasonic wave, often act on 30 ~ 60min and stop 10-30min, repeated action 3 ~ 5 times, liquid is changed 2 ~ 5 times in midway, with 0.05MTris, 1MNaCl, pH is the Tris-NaCl buffered soln cleaning 3 ~ 5 times of 7.5, then is immersed in 2 ~ 3.5MNaCl, in the hypertonic solution of 20 ~ 40mMEDTA, 10 ~ 20h, draining 30 ~ 60min is stirred at 37 DEG C; Animal cartilage is continued to be immersed in pH2.0 ~ 2.5, acetum 2 ~ the 10h of 1000 ~ 5000 parts by volume, filtered through gauze recovery of acetic acid solution, washed with de-ionized water 3 ~ 5 times, with 0.1 ~ 1M, the NaOH solution sofening treatment 10-24h of 1000 ~ 5000 parts by volume, washed with de-ionized water 3 ~ 5 times, lyophilize, obtains the animal cartilage of purifying; Adopted by animal cartilage superfine comminution at low temperature machine to pulverize, preserve stand-by in moisture eliminator;
(2) extraction of non-denatured type II collagen: 100 ~ 200 parts of animal cartilage powder are immersed in the 0.05MTris that 1000 ~ 2000 parts by volume contain 4M Guanidinium hydrochloride, 1MNaCl, pH is in the Tris-NaCl buffered soln of 7.5, stirred at ambient temperature 10 ~ 36h, then the centrifugal 10 ~ 30min of 10000 ~ 20000rpm, reclaim supernatant liquor, with deionized water washing and precipitating thing repeatedly, throw out is immersed in pH2.0 ~ 2.5, the acetum of 3000 ~ 10000 parts by volume, slowly 3 ~ 10h is stirred at constant temperature 4 ~ 10 DEG C, then 1 ~ 4 part of prozyme is added, 24 ~ 72h is rotated at 4 ~ 10 DEG C, obtain enzyme molten animal cartilage II Collagen Type VI solution,
(3) purifying of non-denatured type II collagen: to above-mentioned gained to animal cartilage II Collagen Type VI solution in add the sodium chloride powder that ultimate density is 0.9mol/L, 10 ~ 16h is stirred at 4 ~ 10 DEG C, centrifugal 10 ~ the 30min of 10000 ~ 20000rpm, abandon supernatant liquid, throw out is dissolved in 0.1 ~ 0.5mol/L acetum again, be 6.5 ~ 7.5 by 5 ~ 10MNaOH solution adjust ph, add the sodium chloride powder that ultimate density is 4mol/L, 10 ~ 16h is stirred at 4 ~ 10 DEG C, then the centrifugal 10 ~ 30min of 10000 ~ 20000rpm, abandon supernatant liquid, throw out is dissolved in 0.1 ~ 0.5mol/L acetum again, be 6.5 ~ 7.5 by 5 ~ 10MNaOH solution adjust ph again, add the sodium chloride powder that ultimate density is 4mol/L, 10 ~ 16h is stirred at 4 ~ 10 DEG C, again with the centrifugal 10 ~ 30min of 10000 ~ 20000rpm, take off a layer throw out, so repeatedly saltout 3 ~ 5 times, the precipitation obtained after saltouing is dissolved in 0.1 ~ 0.5mol/L acetum again, take concentration as the acetic acid solution of 0.1 ~ 0.5M is dialyzate dialysis treatment 2-3 days, and freeze-drying, obtains spongy unmodified animal cartilage II Collagen Type VI, obtained animal cartilage II Collagen Type VI, its key property reaches following index request:
Outward appearance: white sponge, the impurity visible without naked eyes and variable color;
Tryptophan analysis: should not tryptophane be contained;
Heavy metal content :≤10 μ g/g (m/m);
Hydroxyproline content: 8.5% (m/m) that should be not less than total protein content;
Liposome :≤1% (m/m);
Ash content :≤2% (m/m);
Foreign protein content :≤1% (m/m);
Cytotoxicity: cell-cytotoxic reaction is not more than 1 grade;
Sterility test: aseptic;
Sensitization test: without delayed hypersensitivity;
Intradermoreaction is tested: primary stimulation index PII<0.4.
2. the preparation method of the non-denatured type II collagen in animal cartilage source as claimed in claim 1, is characterized in that the weight ratio of its three is 3:2:0.5 containing stomach en-, papoid and dextranase in described prozyme.
3. the preparation method of the non-denatured type II collagen in animal cartilage source as claimed in claim 1, it is characterized in that described animal cartilage be fresh, can trace to the source, can be pig, ox, chicken, any one cartilage of sheep.
4. the preparation method of the non-denatured type II collagen in animal cartilage source as claimed in claim 1, it is characterized in that described grease-removing agent formula is: 4.0g/L sodium polyphosphate, 3.5g/LEDTA, 18g/L Sodium dodecylbenzene sulfonate, 10g/L DC11 trolamine, 15g/L peregal.
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| CN110082486A (en) * | 2019-05-14 | 2019-08-02 | 中国农业科学院农业质量标准与检测技术研究所 | The method traced to the source using the stable isotope of ossein the mutton sheep place of production |
| CN111662375A (en) * | 2020-06-11 | 2020-09-15 | 湖北和格复合骨胶原生物科技有限公司 | Preparation method for extracting non-denatured type II collagen from animal cartilage |
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