CN105331662A - Non-denatured II type collagen of animal cartilage source and preparation method for non-denatured II type collagen - Google Patents

Abstract

The invention discloses a preparation method of undenatured type II collagen derived from animal cartilage, which is characterized in that fresh and traceable animal cartilage is used as raw material, through repeated freezing and thawing, ultrasonic degreasing, decellularization of hypertonic fluid, acid-base softening, etc. The process purifies the cartilage, and then undergoes processes such as guanidine hydrochloride desugar, pickling, compound enzyme treatment, multiple salting out, multiple centrifugal purification, membrane dialysis, and freeze-drying to obtain spongy undenatured type II collagen. The high-purity and high-yield type II collagen obtained by this method retains the complete triple helical structure of collagen, has high biological activity, stable structure and easy storage, and is beneficial to the adhesion, growth and proliferation of chondrocytes, and can be used in the production of medical biomaterials. preparation.

Description

A kind of undenatured type II collagen derived from animal cartilage and its preparation method

technical field

The invention relates to a method for preparing undenatured type II collagen derived from animal cartilage, belonging to the field of biomedical material preparation.

Background technique

Type II collagen is one of the main components of the cartilage matrix. It is similar to type I collagen and is a fiber-forming collagen. It consists of three identical αI (II) chains to form a triple helical domain. Its molecules do not contain tryptophan residues. The content of tyrosine residues is also less, so the antigenicity of its molecules is lower, especially the type II collagen produced by enzymatic method, after removing the telopeptide of the molecular chain, its immunogenicity is lower. Type II collagen molecules in cartilage are bridged and cross-linked by covalent bonds to form a stable three-dimensional network structure. Hydroxylysine, which is rich in type II collagen, is covalently bonded with polysaccharides, which further stabilizes the natural state of type II collagen. Spatial structure, which is beneficial to protect the structure and biological activity of cartilage, and maintain the mechanical properties and mechanical stability of natural cartilage. At the same time, the spatial structure of natural type II collagen determines its low solubility in water, which is not conducive to the separation and purification of type II collagen. Therefore, it is particularly important to maintain the triple helical structure of collagen well during the extraction process, so as not to further decompose the collagen and lose its physiological activity due to excessive treatment. So far, the extraction and preparation methods of type II collagen are mainly acid method, alkali method, enzymatic method, combination method, neutral salt method and hot water extraction method. The molecules of type II collagen obtained by each method are There are significant differences in structure and performance. Among them, type II collagen obtained by alkali method and hot water extraction method has low molecular weight and wide distribution, and does not have biological activity; Molecular telopeptides may cause type II collagen to have certain immunogenicity, affecting its biological application, and the extraction rate of the obtained collagen is too low, which is not easy for industrial production; type II collagen prepared by enzymatic method can completely retain its molecular structure, The extraction rate is also relatively high, making it most suitable for large-scale production. However, there is a contradiction between the purity and yield of collagen in the preparation of type II collagen by enzymatic method. If the yield is high, the purity will not meet the requirements. Therefore, it is very important to extract and prepare type II collagen with high yield and high purity. important.

Contents of the invention

The purpose of the present invention is to provide a method for preparing undenatured type II collagen of animal cartilage origin in view of the deficiencies in the prior art, which is characterized in that the undenatured type II collagen prepared by the method has high yield, high purity and high biological activity , the structure is stable and easy to preserve, it is beneficial to the adhesion, growth and proliferation of chondrocytes, and can be used for the preparation of medical biomaterials.

To achieve the above object, the present invention adopts the following technical solutions:

(1) Pretreatment of animal cartilage: Take 100-200 copies of fresh and traceable animal cartilage, remove residual bones and muscles manually with scissors, then wash repeatedly with saline in a stainless steel drum for 3-5 times, and drain for 30-60 minutes ;Put the animal cartilage in a quick-freezing refrigerator at -40-80°C for 5-10 times, then soak it in 1000-10000 parts by volume of degreasing agent, accompanied by intermittent power of 50-100W ultrasonic waves, every Act for 30-60 minutes and stop for 10-30 minutes, repeat the action for 3-5 times, change the medium 2-5 times in the middle, wash with 0.05MTris, 1MNaCl, Tris-NaCl buffer solution with pH 7.5 for 3-5 times, and then soak it in In the hypertonic solution of 2-3.5M NaCl, 20-40mM EDTA, stir at 37°C for 10-20h, drain for 30-60min; continue to soak the animal cartilage in pH2.0-2.5, 1000-5000 volume parts of acetic acid solution for 2-10h , filtered through gauze to recover the acetic acid solution, washed with deionized water for 3 to 5 times, softened with 0.1 to 1M, 1000 to 5000 parts by volume of NaOH solution for 10 to 24 hours, washed with deionized water for 3 to 5 times, and freeze-dried to obtain purified Animal cartilage; the animal cartilage is pulverized with a low-temperature ultrafine pulverizer, and stored in a desiccator for later use;

(2) Extraction of undenatured type II collagen: Soak 100-200 parts of animal cartilage powder in 1000-2000 parts by volume of 0.05M Tris containing 4M guanidine hydrochloride, 1M NaCl, Tris-NaCl buffer solution with a pH of 7.5, and stir at room temperature 10～36h, then centrifuge at 10000～20000rpm for 10～30min, recover the supernatant, wash the precipitate repeatedly with deionized water, soak the precipitate in pH2.0～2.5, 3000～10000 parts by volume of acetic acid solution, at constant temperature Stir slowly at ~10°C for 3-10 hours, then add 1-4 parts of compound enzyme, and rotate at 4-10°C for 24-72 hours to obtain an enzymatically dissolved animal cartilage type II collagen solution;

(3) Purification of undenatured type II collagen: Add sodium chloride powder with a final concentration of 0.9mol/L to the above-obtained animal cartilage type II collagen solution, stir at 4-10°C for 10-16h, 10000-20000rpm Centrifuge for 10-30 minutes, discard the supernatant, dissolve the precipitate in 0.1-0.5mol/L acetic acid solution again, adjust the pH value to 6.5-7.5 with 5-10M NaOH solution, and add sodium chloride powder with a final concentration of 4mol/L , stirred at 4-10°C for 10-16h, then centrifuged at 10000-20000rpm for 10-30min, discarded the supernatant, dissolved the precipitate in 0.1-0.5mol/L acetic acid solution again, and adjusted the pH value with 5-10M NaOH solution 6.5~7.5, add sodium chloride powder with a final concentration of 4mol/L, stir at 4~10°C for 10~16h, centrifuge again at 10000~20000rpm for 10~30min, remove the lower layer of sediment, and repeat the salting out for 3~5 times ; Dissolve the precipitate obtained after salting out in 0.1-0.5mol/L acetic acid solution, dialyze with 0.1-0.5M acetic acid solution as the dialysate for 2-3 days, and freeze-dry to obtain spongy undenatured animals Cartilage type II collagen; the prepared animal cartilage type II collagen, its key properties meet the following index requirements:

Appearance: white sponge, no impurities and discoloration visible to the naked eye;

Tryptophan analysis: should not contain tryptophan;

Heavy metal content: ≤10μg/g(m/m);

Hydroxyproline content: not less than 8.5% (m/m) of the total protein content;

Liposome: ≤1% (m/m);

Ash content: ≤2%(m/m);

Miscellaneous protein content: ≤1% (m/m);

Cytotoxicity: the cytotoxic reaction is not greater than grade 1;

Sterility test: sterile;

Sensitization test: no delayed hypersensitivity reaction;

Intradermal test: primary irritation index PII<0.4.

In the above preparation method, step (1) is a medium and low temperature ultrafine pulverizer, produced by Beijing Zhongke Haoyu Technology Development Co., Ltd.; the ultrasonic time in step (1) varies depending on the ultrasonic power used; The activity unit of pepsin in the compound enzyme is 3000 units/g, the activity unit of papain is 8 million units/g, and the activity unit of dextranase is 50 units/g; the preparation process is all completed at 4-10°C.

This patented technology pre-treats animal cartilage through repeated freezing and thawing, ultrasonic degreasing, decellularization of hypertonic fluid, acid-alkali alternate softening, etc., which can effectively remove the non-collagen components of cartilage, and further loosen the collagen fibers in animal cartilage. The gap between the collagen fibers of animal cartilage is increased, which is very conducive to the full penetration and action of biological enzymes in the extraction process, and can effectively improve the purity and yield of collagen; the synergistic effect of the components of the compound enzyme can maximize the removal of collagen telopeptide, reducing its immunogenicity.

Undenatured Type II Collagen has the following uses:

1. Used as raw materials for biomedical materials, such as raw materials for the preparation of drug controlled release carriers, hemostatic materials, dura mater repair materials, tissue engineering scaffolds, tissue guiding materials, plastic surgery materials, etc.;

2. Used as raw materials for cosmetic materials, such as skin creams, hair conditioners, etc.;

3. Used as raw materials for food industry materials, such as raw materials for health care materials and beverages;

4. Others, such as cell culture, bioreactor monomer membrane, etc.

Compared with the prior art, this technology has the following advantages:

(1) The present invention uses ultrasonic waves in the purification process of animal cartilage, fully exerts the cavitation effect, mechanical effect, thermal effect and other effects of ultrasonic waves, destroys the cell membrane of cells in cartilage, and is beneficial to further degreasing and decellularization of cartilage ;

(2) The present invention uses compound enzymes to extract type II collagen, and the synergistic effect between the components of the compound enzymes is extremely beneficial to the removal of non-collagen components, miscellaneous cells and collagen telopeptides in animal cartilage, and can effectively increase the yield of collagen and purity;

(3) In the preparation process of type II collagen described in the present invention, the animal cartilage is firstly purified through processes such as repeated freezing and thawing, ultrasonic degreasing, decellularization of hypertonic fluid, acid-alkali alternate softening, etc., to fully remove the collagen in the cartilage. Non-collagen components loosen the collagen fibers in cartilage, which can effectively improve the yield and purity of collagen;

(4) The type II collagen prepared by the present invention has rich materials, low cost, advanced and reliable technology, large output, and is suitable for mass production.

detailed description

The present invention will be specifically described below by implementing it. It is necessary to point out that this embodiment is only used to further illustrate the present invention, and can not be interpreted as limiting the protection scope of the present invention. Make non-essential improvements and adjustments to the content.

Example 1

(1) Pre-treatment of animal cartilage: Take 100 parts of fresh and traceable animal cartilage, manually remove residual bones and muscles with scissors, then wash with normal saline for 3 times, drain for 30 minutes; put animal cartilage in a -40℃ quick-freezing Repeated freezing and thawing in the refrigerator for 5 times, then soaking it in 1000 volume parts of degreasing agent in a stainless steel drum, accompanied by intermittent power of 50W ultrasonic wave every 60 minutes and stopping for 30 minutes, repeated treatment 5 times, and changing the liquid 5 times in the middle, Wash 3 times with 0.05M Tris, 1M NaCl, Tris-NaCl buffer solution with pH 7.5, then soak it in the hypertonic solution of 2M NaCl, 20mM EDTA, stir at 37°C for 10h, drain for 30min; continue to soak the animal cartilage in pH2. 0, 1000 parts by volume of acetic acid solution for 2 hours, filtered through gauze to recover the acetic acid solution, washed 3 times with deionized water, softened with 0.1M, 1000 parts by volume of NaOH solution for 10 hours, washed 3 times with deionized water, and freeze-dried to obtain purified Animal cartilage; the animal cartilage is pulverized with a low-temperature ultrafine pulverizer, and stored in a desiccator for later use;

(2) Extraction of undenatured type II collagen: Soak 100 parts of animal cartilage powder in 1000 volume parts of 0.05M Tris containing 4M guanidine hydrochloride, 1M NaCl, Tris-NaCl buffer solution with a pH of 7.5, stir at room temperature for 10h, then 10000rpm Centrifuge for 30 minutes, recover the supernatant, wash the precipitate repeatedly with deionized water, soak the precipitate in pH 2.0, 3000 parts by volume of acetic acid solution, stir slowly at a constant temperature of 4°C for 3 hours, then add 1 part of compound enzyme, 4 Rotate at ℃ for 72 hours to obtain enzymatically dissolved animal cartilage type II collagen solution;

(3) Purification of undenatured type II collagen: add sodium chloride powder with a final concentration of 0.9 mol/L to the obtained animal cartilage type II collagen solution, stir at 4°C for 10 h, centrifuge at 10,000 rpm for 30 min, and discard the supernatant Solution, dissolve the precipitate in 0.1mol/L acetic acid solution again, adjust the pH value to 6.5 with 5M NaOH solution, add sodium chloride powder with a final concentration of 4mol/L, stir at 4°C for 10h, then centrifuge at 10000rpm for 30min, discard the upper layer Clear liquid, dissolve the precipitate in 0.1mol/L acetic acid solution again, adjust the pH value to 6.5 with 5M NaOH solution, add sodium chloride powder with a final concentration of 4mol/L, stir at 4°C for 10h, and centrifuge again at 10000rpm for 30min , remove the lower layer of precipitate, so repeated salting out 3 times; redissolve the precipitate obtained after salting out in 0.1mol/L acetic acid solution, use 0.1M acetic acid solution as the dialysate for dialysis treatment for 2 days, and freeze-dry to obtain Spongy undenatured animal cartilage type II collagen.

Example 2

(1) Pre-treatment of animal cartilage: Take 150 parts of fresh and traceable animal cartilage, manually remove residual bones and muscles with scissors, then repeatedly wash 4 times with normal saline in a stainless steel drum, and drain for 40 minutes; put the animal cartilage into Repeated freezing and thawing 8 times in a quick-freezing refrigerator at -50°C, then soaking it in 4500 volume parts of degreasing agent, accompanied by intermittent power of 60W ultrasonic wave every 40 minutes and stopping for 20 minutes, repeated action 4 times, and changing the liquid 3 times in the middle, Wash 4 times with 0.05M Tris, 1M NaCl, Tris-NaCl buffer solution with pH 7.5, then soak it in the hypertonic solution of 3M NaCl, 30mM EDTA, stir at 37°C for 15h, drain for 40min; continue to soak the animal cartilage in pH2. 2, 4500 parts by volume of acetic acid solution for 8 hours, filtered through gauze to reclaim the acetic acid solution, washed 4 times with deionized water, softened with 0.5M, 4500 parts by volume of NaOH solution for 18 hours, washed 4 times with deionized water, freeze-dried to obtain purified Animal cartilage; the animal cartilage is pulverized with a low-temperature ultrafine pulverizer, and stored in a desiccator for later use;

(2) Extraction of undenatured type II collagen: Soak 150 parts of animal cartilage powder in 1500 parts by volume of 0.05M Tris containing 4M guanidine hydrochloride, 1M NaCl, Tris-NaCl buffer solution with a pH of 7.5, and stir at room temperature for 10-36 hours. Then centrifuge at 15,000 rpm for 20 minutes, recover the supernatant, wash the precipitate repeatedly with deionized water, soak the precipitate in pH 2.2, 4,500 parts by volume of acetic acid solution, stir slowly at a constant temperature of 5°C for 5 hours, and then add 3 parts of compound enzyme , and rotated at 5°C for 36 hours to obtain an enzymatically soluble animal cartilage type II collagen solution;

(3) Purification of undenatured type II collagen: add sodium chloride powder with a final concentration of 0.9 mol/L to the obtained animal cartilage type II collagen solution, stir at 5°C for 14 h, centrifuge at 15,000 rpm for 20 min, and discard the supernatant Solution, dissolve the precipitate in 0.2mol/L acetic acid solution again, adjust the pH value to 7.0 with 8M NaOH solution, add sodium chloride powder with a final concentration of 4mol/L, stir at 5°C for 12h, then centrifuge at 15000rpm for 20min, discard the upper layer Clear liquid, dissolve the precipitate in 0.2mol/L acetic acid solution again, adjust the pH value to 7.0 with 8M NaOH solution, add sodium chloride powder with a final concentration of 4mol/L, stir at 5°C for 12h, and centrifuge again at 15000rpm for 20min , remove the lower layer of precipitate, so repeated salting out 4 times; redissolve the precipitate obtained after salting out in 0.2mol/L acetic acid solution, use 0.2M acetic acid solution as the dialysate for dialysis treatment for 2 days, and freeze-dry to obtain Spongy undenatured animal cartilage type II collagen.

Example 3

(1) Pre-treatment of animal cartilage: Take 200 parts of fresh and traceable animal cartilage, manually remove residual bones and muscles with scissors, then wash with normal saline for 5 times, drain for 60 minutes; put animal cartilage in a -80°C freezer Freeze and thaw repeatedly in the refrigerator for 10 times, then soak it in 10,000 volume parts of degreasing agent in a stainless steel drum, with an intermittent power of 100W ultrasonic wave every 30 minutes and stop for 10 minutes, repeat the action 3 times, change the liquid twice in the middle, Wash 5 times with 0.05M Tris, 1M NaCl, Tris-NaCl buffer solution with a pH of 7.5, then soak it in a hypertonic solution of 3.5M NaCl, 40mM EDTA, stir at 37°C for 20 hours, and drain for 60 minutes; continue to soak the animal cartilage in pH 2 .5,5000 volume parts of acetic acid solution 2h, gauze filtration reclaims acetic acid solution, deionized water washes 5 times, with 1M, the NaOH solution softening treatment of 5000 volume parts 24h, deionized water washes 5 times, freeze-dries, obtains purified Animal cartilage; the animal cartilage is pulverized with a low-temperature ultrafine pulverizer, and stored in a desiccator for later use;

(2) Extraction of undenatured type II collagen: Soak 200 parts of animal cartilage powder in 2000 volume parts of 0.05M Tris containing 4M guanidine hydrochloride, 1M NaCl, Tris-NaCl buffer solution with a pH of 7.5, stir at room temperature for 36h, then 20000rpm Centrifuge for 10 minutes, recover the supernatant, wash the precipitate repeatedly with deionized water, soak the precipitate in pH 2.5, 10,000 parts by volume of acetic acid solution, stir slowly at a constant temperature of 10°C for 10 hours, then add 4 parts of compound enzyme, 10 Rotate at ℃ for 24 hours to obtain enzymatically dissolved animal cartilage type II collagen solution;

(3) Purification of undenatured type II collagen: add sodium chloride powder with a final concentration of 0.9 mol/L to the obtained animal cartilage type II collagen solution, stir at 10°C for 16 h, centrifuge at 20,000 rpm for 10 min, and discard the supernatant Solution, dissolve the precipitate in 0.5mol/L acetic acid solution again, adjust the pH value to 7.5 with 10M NaOH solution, add sodium chloride powder with a final concentration of 4mol/L, stir at 10°C for 16h, then centrifuge at 20000rpm for 10min, discard the upper layer Clear liquid, dissolve the precipitate in 0.5mol/L acetic acid solution again, adjust the pH value to 7.5 with 10M NaOH solution, add sodium chloride powder with a final concentration of 4mol/L, stir at 10°C for 16h, and centrifuge again at 20000rpm for 10min , remove the lower layer of precipitate, and repeat the salting out 5 times; the precipitate obtained after salting out is redissolved in 0.5mol/L acetic acid solution, and the acetic acid solution with a concentration of 0.5M is used as the dialysate for dialysis treatment for 3 days, freeze-dried, Obtain spongy undenatured animal cartilage type II collagen.

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.