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

A cartilage and animal technology, which is applied in the field of preparation of undenatured type II collagen, can solve the problems of insufficient purity, low extraction rate of collagen, and difficulty in industrial production, etc., to achieve improved yield and purity, advanced and reliable technology, and material extraction rich effect

Inactive Publication Date: 2016-02-17
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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, which is 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

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] (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...

Embodiment 2

[0037] (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, sof...

Embodiment 3

[0041] (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 5...

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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 [0001] 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 [0002] 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K1/30
Inventor 但年华刘新华但卫华
Owner SICHUAN UNIV
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