CN106319015A - Preparation method of II-type collagen with biological activity in shark cartilage - Google Patents

Abstract

The invention discloses a preparation method of biologically active type II collagen in shark cartilage. The steps are as follows: after the shark cartilage is processed, it is freeze-dried, crushed into 80-200 mesh powder, treated with sodium hydroxide, hydrolyzed with pepsin under restricted conditions, salted out with sodium chloride, dialysis, freeze-dried and other processes to obtain a relatively pure High type II collagen. The method of the invention is easy to operate and easy to extract, has higher safety and wider application range, and the biological activity of the obtained type II collagen is not destroyed, and has excellent characteristics as a biological functional substance.

Description

A preparation method of biologically active type II collagen in shark cartilage

technical field

The invention belongs to the field of marine natural products, and relates to a preparation method of biologically active type II collagen in shark cartilage.

Background technique

Type II collagen exists in mammalian cartilage in the superhelical structure of procollagen homotrimer, which is composed of three identical α1(Ⅱ) chains. During the synthesis process, the modification of procollagen α chain is through proline Acyl and lysyl hydroxylases hydroxylate proline and lysine in cells, glycosylate specific hydroxylysine, combine chains and generate intra-chain disulfide bonds, thus forming triple chains spiral structure. Type II collagen is only found in cartilage tissue, which constitutes 60% of the dry weight of cartilage, and is abundant in mammalian hyaline cartilage, accounting for about 80% of the total protein content of cartilage tissue. The two main components of cartilage are fibrous type II collagen and large molecular weight proteoglycans, which can protect tissues. Most of the physiological characteristics of cartilage are dependent on the intact type II collagen network.

Type II collagen is initially synthesized in the form of precursors in fibroblasts of cartilage connective tissue, and then spliced into procollagen IIA and procollagen IIB. The different molecular forms of type II collagen are due to the half of the amino terminal propeptide. Formed by selective cleavage of cystine-rich regions. Procollagen IIA has a cysteine-rich region at the N-terminus, called type II procollagen A amino-terminal propeptide, which is mainly found in pre-cartilage tissue fibroblasts and embryonic tissues, while the N-terminus of procollagen IIB lacks this A cysteine-rich region predominantly found in adult mammalian cartilage fibroblasts. The type II collagen precursor cuts off the amino-terminal propeptide, and after being modified by hydroxylation, it is connected into a triple helix structure through a disulfide bond and secreted to the outside of the cell to become a structural protein in the cartilage matrix.

As a typical structural protein, type II collagen contains most of the common amino acids, including high content of glycine and hydroxyproline, but does not contain tryptophan or other amino acids. The spatial superhelical structure formed by the three peptide chains of collagen based on amino acids is closely related to the repeated Gly-X-Y tripeptide units of each peptide chain. The spatial position of glycine can optimize the folding conformation of the polypeptide chain, and glycine is the only amino acid suitable for the internal position of the peptide chain folding, resulting in the repetition of (Gly-X-Y)n amino acid sequence, the most common tripeptide unit Gly-Pro-Hyp contributes to The stability of the triple helix. The thermal denaturation temperature of collagen isolated from shark cartilage by Phanat et al. was 34.56-36.73°C. Chen Shenru and others extracted collagen from gray star shark cartilage, cut the shark cartilage into small pieces regularly, and took about 5 days for decalcification and washing.

Type II collagen is generally isolated from the cartilage of terrestrial organisms such as pigs, sheep and chickens. The extraction method of type II collagen is mainly pepsin hydrolysis, but the pretreatment of cartilage powder to remove non-collagen components and purification steps are the special features of the present invention. The present invention uses 0.1mol/L NaOH and 0.5mol/L EDTA The non-collagen components in the cartilage are removed, the potential safety hazards of guanidine hydrochloride residues are eliminated, the safety is higher, and the application range is wider.

Contents of the invention

The technical problem to be solved by the present invention is to provide a production method of biologically active type II collagen, the extraction process of the collagen is relatively simple, the safety is higher, and the application range is wider.

A preparation method with bioactive type II collagen in shark cartilage, comprising the steps of:

(1) Obtain shark cartilage material, cut the obtained cartilage into 3-5mm thin slices, and freeze-dry the chopped cartilage slices; after freeze-drying and crushing, the extraction rate of collagen can be maximized and some biologically active functions can be maintained .

(2) pulverize the freeze-dried cartilage into 80-200 mesh powder, and carry out the impurity removal process;

(3) Type II collagen was hydrolyzed and extracted by pepsin to obtain a mixed solution containing type II collagen; the extraction efficiency of type II collagen was higher in acidic environment.

(4) After salting out, dialysis and freeze-drying, type II collagen with biological activity is obtained.

Further, the shark cartilage material is selected from blue shark cartilage. Slightly higher extraction efficiency than other shark cartilage.

Further, the impurity removal process in the step (2) is specifically as follows: Shark cartilage powder is mixed with 0.1-0.2mol/L NaOH1:5-15 volume ratio, fully stirred, the lye is replaced once every 2-3 hours, and stirred After the end, wash repeatedly with deionized water until the pH is 7.0-7.6, then use 0.5-1mol/L, pH7.0-7.6EDTA solution to continuously stir to remove minerals, replace the solution every 8-10h, and use 20 - 30 times the volume of deionized water was continuously stirred for 10-20 minutes, 2-5 times in total, and finally the shark cartilage powder that was swelled after removing impurities was obtained. The conditions for the existence of type II collagen are relatively moderate, which is conducive to the maintenance of its biological activity.

Further, the specific steps of step (3) hydrolyzing and extracting type II collagen with pepsin are as follows: mix the swollen shark cartilage powder after removing impurities with 1-2% pepsin solution prepared with 0.5-1mol/L acetic acid 1:10-20 volume ratio mixing, after full stirring, centrifuge at 6000-10000rpm, take the supernatant, adjust the pH to 7.4-7.6 with NaOH; add NaCl for salting out, and stir to fully precipitate type II collagen, 6000 Centrifuge at -10000rpm to obtain precipitate; dialyze, use 0.5-1mol/L acetic acid for precipitation to make it just dissolve, dialyze with 20-30 times volume of 0.1-0.2mol/L acetic acid for 12-36h, and then use 20-30 times volume to remove Dialyze with ionized water for 24-72 hours; the dialysate should be freeze-dried and stored.

Further, all operations except freeze-drying were carried out at 3-5°C.

Further, all operations except freeze-drying were carried out at 4°C to maintain the biological activity of type II collagen.

The beneficial effects of the present invention are:

1. The present invention freeze-dries the raw material and then pulverizes it, which not only maintains its biological activity, but also provides convenience for the subsequent processing of the raw material.

2. The technical scheme of the present invention is simple and easy to implement, with strong standardization and high output.

3. In the present invention, the extraction process of type II collagen is relatively simple and easy to scale up.

4. In the present invention, the treatment process of the cartilage powder is more original, simple and easy to implement, with little pollution and low energy consumption.

detailed description

Example 1

1. Take 10kg of shark cartilage. Impurities such as residual meat and periosteum were manually removed, washed with normal saline 3 times and dried in the air.

2. Cut the cartilage into 3mm slices, and freeze-dry them in a freeze dryer at -25°C and 10Pa.

3. Crush the cartilage into 200 mesh powder.

4. Mix shark cartilage powder with 0.1mol/L NaOH in a volume ratio of 1:10, stir thoroughly, replace the lye every 2 hours, wash repeatedly with deionized water after stirring, until the pH is 7.0, and use 0.5mol/L L, pH 7.4 EDTA solution was continuously stirred to remove mineral matter, and the solution was replaced every 8 hours. After the stirring was completed, it was continuously stirred for 10 minutes with 20 times the volume of deionized water, for a total of 3 times, and finally the swelled shark cartilage powder was obtained after removing impurities.

5. Mix the swollen shark cartilage powder with 1% pepsin solution prepared with 0.5mol/L acetic acid at a volume ratio of 1:10. After fully stirring, centrifuge at 6000-10000rpm, take the supernatant, and dissolve it with NaOH Adjust the pH to 7.5; add NaCl to 2.6mol/L for salting out, and stir to fully precipitate type II collagen, centrifuge at 8000rpm to obtain a precipitate; dialyze, use 0.5mol/L acetic acid for precipitation to make it just dissolve, and use 25 times the volume of Dialyze with 0.1mol/L acetic acid for 24 hours, and then dialyze with 25 times the volume of deionized water for 48 hours; the dialysate is freeze-dried and stored.

Obtained 1.16kg type Ⅱ collagen, the purity of type Ⅱ collagen was determined to be 93.4%, the molecular weight was 130kD, the α1 chain and its dimer were shown on SDS-PAGE, and the hydroxyproline and glycine in the extracted type Ⅱ collagen The content of Gly reaches 7.09%, and Gly residues account for more than 30% of the total amino acid residues. Blue shark type II collagen has the characteristics of amino acid residues (Gly-Xaa-Yaa), which is in line with the characteristics of collagen.

After testing, the heat distortion temperature reached 41°C.

As determined by the antioxidant kit, the scavenging rate of type II collagen to DPPH free radicals is 21.85%.

Comparative example 1

1. Take 10kg of shark cartilage. Impurities such as residual meat and periosteum were manually removed, washed with normal saline 3 times and dried in the air.

2. Cut the cartilage into 3mm slices.

3. Crush the cartilage into 200 mesh powder.

4. Mix shark cartilage powder with 0.1mol/LNaOH in a volume ratio of 1:10, stir fully, replace the lye every 2 hours, wash repeatedly with deionized water after stirring, until the pH is 7.0, use 0.5mol/L , the pH7.4 EDTA solution was continuously stirred to remove minerals, and the solution was replaced every 8 hours. After the stirring was completed, 20 times the volume of deionized water was continuously stirred for 10 minutes, a total of 3 times, and finally the shark cartilage powder that was swelled after removing impurities was obtained.

5. Mix the swollen shark cartilage powder with 1% pepsin solution prepared with 0.5mol/L acetic acid at a volume ratio of 1:15. After fully stirring, centrifuge at 6000-10000rpm, take the supernatant, and dissolve it with NaOH Adjust the pH to 7.5; add NaCl to 2.6mol/L for salting out, and stir to fully precipitate type II collagen, centrifuge at 8000rpm to obtain a precipitate; dialyze, use 0.5mol/L acetic acid for precipitation to make it just dissolve, and use 25 times the volume of Dialyze with 0.1mol/L acetic acid for 24 hours, and then dialyze with 25 times the volume of deionized water for 48 hours; the dialysate is freeze-dried and stored.

1.09kg type II collagen was obtained, and the purity of type II collagen was determined to be 91.7%.

As determined by the antioxidant kit, the scavenging rate of type II collagen to DPPH free radicals is 16.56%.

From the above examples, it can be concluded that the extraction rate of collagen can be maximized and part of its biological activity can be maintained by crushing shark bone after freeze-drying. Type II collagen has a scavenging rate of about 20% for DPPH free radicals, which is much higher than that of animal protein extracts such as scallop meat, and the extraction rate is about 10%. Compared with the same type of collagen extraction method, this method The extraction rate is higher, and the heat distortion temperature is increased to 41°C, which increases the potential scope of application of type II collagen.

Claims (5)

1. shark cartilage has a preparation method for biological activity typeⅡ Collagen, comprises the steps:

(1) obtain cartilage material, the cartilage of acquisition is processed and after cleaning, is cut into 3-5mm thin slice, by the cartilage thin slice of chopping Lyophilization；

(2) Cartilage powder after lyophilization is broken into the powder of 80-200 mesh again；

(3) being mixed with 0.1-0.2mol/L NaOH 1:5-15 volume ratio by shark cartilage powder, be sufficiently stirred for, every 2-3 is little Alkali liquor of Shi Genghuan, stirring uses deionized water cyclic washing after terminating, until pH is 7.0-7.6, then with 0.5-1mol/L, PH7.0-7.6EDTA solution continuous stirring removes mineral, and every 8-10h changes a solution, stirs after terminating with 20-30 times of body Long-pending deionized water continuous stirring 10-20min, 2-5 time altogether, the shark cartilage powder of swollen profit after finally obtaining the removal of impurity；

(4) by the shark cartilage powder of the swollen profit of step (3) gained and the 1-2% pepsin liquid being made into 0.5-1mol/L acetic acid Mixing with 1:10-20 volume ratio, after being sufficiently stirred for, 6000-10000rpm is centrifuged, and takes supernatant, is adjusted to by pH with NaOH 7.4-7.6；Adding NaCl to saltout, and stirring makes typeⅡ Collagen fully separate out, 6000-10000rpm is centrifugal to obtain precipitation；Thoroughly Analysis, the 0.5-1mol/L acetic acid of precipitation is allowed to just dissolve, with the 0.1-0.2mol/L acetic acid dialysis 12-of 20-30 times of volume 36h, then dialyse 24-72h with the deionized water of 20-30 times of volume；Preservation after dialysis solution lyophilization, obtains having biological activity TypeⅡ Collagen.

Shark cartilage the most according to claim 1 has the preparation method of biological activity typeⅡ Collagen, its feature It is: described cartilage material is selected from blue shark cartilage.

Shark cartilage the most according to claim 1 has the preparation method of biological activity typeⅡ Collagen, its feature It is: described lyophilization is carried out under the conditions of-30～-20,5-20Pa.

Shark cartilage the most according to claim 1 has the preparation method of biological activity typeⅡ Collagen, its feature It is: in addition to lyophilization, all operations is all carried out under the conditions of 3-5 DEG C.

Shark cartilage the most according to claim 4 has the preparation method of biological activity typeⅡ Collagen, its feature It is: in addition to lyophilization, all operations condition is carried out under the conditions of 4 DEG C.