CN101307093A

CN101307093A - Method for separating and purifying collagen for biopharmaceutical use from sharkskin

Info

Publication number: CN101307093A
Application number: CNA2007100405900A
Authority: CN
Inventors: 周培根; 缪辉南; 吴文惠; 何培民; 蔡春尔; 李燕
Original assignee: Shanghai Ocean University
Current assignee: Shanghai Ocean University
Priority date: 2007-05-14
Filing date: 2007-05-14
Publication date: 2008-11-19
Anticipated expiration: 2027-05-14
Also published as: CN101307093B

Abstract

A method for obtaining collagen for biomedical use from shark skin through separation and purification adopts the raw material of the shark skin. The method has the following steps: steeping the shark skin in low-concentration phosphate buffer solution with a PH value of 7.4-7.6 to flush away squamae and residual flesh, chopping the shark skin in small pieces, and steeping and flushing the shark skin with deionized water for 3-5 times; steeping the clean shark skin in medium-concentration phosphate buffer solution which is five folds of the raw material amount with a PH value of 7.4-7.6 for 70-75 hours, then stirring the pulp evenly for 15 minutes with a temperature of 4 DEG C and a rotating speed of 10000rpm, and centrifuging the pulp for 15 minutes and taking supernatant; depositing and repeating the former step, mixing supernatant, adding ammonium sulphate to a saturation degree of 80 percent, a temperature of 4 DEG C and a rotating speed of 8000rpm, and centrifuging for 15 minutes and retaining deposit; dissolving the deposit in medium-concentration phosphate buffer solution with a PH value of 7.0; chromatographically fractionating the solution with G-75, 100, 150 glucan, collecting albumen digesting liquid with a temperature of 4 DEG C and a rotating speed of 10000rpm, centrifuging for 15 minutes, concentrating supernatant, and obtaining the collagen for biomedical use after freeze drying.

Description

From the method for shark skin separation and purification biological medicine with collagen protein

Technical field:

The present invention relates to the biomedical method of a kind of separation and purification with collagen protein, particularly relate to a kind of from shark skin the biomedical method of separation and purification with collagen protein.

Background technology:

Collagen protein or title collagen are by a kind of biological polymer of zooblast synthetic, extensively be present in animal bone, pin, cartilage and skin and other reticular tissue, account for 1/3 of Mammals total protein, it is the extracellular matrix composition that body weight for humans is wanted, collagen has good biological characteristics, can be used as the upholder of tissue, pair cell, tissue and even organ exercise normal function and reparation has great effect to wound.The utilization to collagen protein both at home and abroad mainly is divided into two big classes: the one, be conceived to the macro physical performance of collagen protein uniqueness, and promptly fibre property is used for printing paper egative film, weaving, papermaking etc.; The 2nd, be conceived to its internal performance, it is the biological function of collagen, be used for health care of food, feed, cosmetics, medical material etc., collagen has important biological property, the mechanical property height, promote the cell growth, hemostasis, biocompatibility and biological degradability, can be used as skin burn, the repairing of scalding, treatment and nursing, can alleviate burn, the degree of scalding, stop to infect, it burns in treatment, the scald aspect plays important effect, it can be processed into " artificial skin ", directly spread on burning, the skin surface of scald, especially for big area, the degree of depth is heavy patient more than the III level, Human-like Collagen can be used as the artificial blood vessel, the repairing of practical vascular graft and human body damaged tissue, for example because of burst disease, gastrorrhagia, perforation, collagen protein can be in conjunction with the antibiosis rope, the short living factor of cell etc. can be repaired damaged tissue, collagen protein can with bone meal, being used for limbs after the bone protein combination extends, collagen protein can be made artificial organs, schrittmacher, the charges of human body viscera etc., collagen protein also is a kind of novel biological fermentation culture medium, contain the amino acid A wide selection of colours and designs, unique effects with microorganism culturing is that bacteriology is cultivated, one of optimum feed stock of biological fermentation culture medium.In addition, by the material of collagen preparation, can be spread to the deep layer of skin, people's skin is had good trophism, owing to contain a large amount of hydroxyls in its molecule, it has goodish moisture-keeping function simultaneously.In 2003 were in great demand the skin care products comparation and assessment most, collagen protein was most popular according to investigations, and collagen protein is for many years popular abroad, and American-European many makeup research and development centres release and contain collagen protein composition skin care products, and the present output value of global collagen protein is above 4,000,000,000 dollars.Extract the purifying aspect about collagen protein, though method is different, but great majority are from squid, extract in the skin of small fish such as cod and domestic animal, the contained collagen protein amount of small fish itself is few, raw material is limited, and this type of albumen solubility that extracts from domestic animal skin is low, and be difficult for being accepted by raw material by the many trade organization in the world with the bovine, the step of extracting collagen protein at present comprises extraction, separate and purification, extraction is divided into acid system again, alkaline process neutral sulfity process and enzyme process etc., technology is loaded down with trivial details, also useful gene engineering method prepares albumen, but has increased cost simultaneously again, because these defectives that above-mentioned these isolation technique exist, the feasible collagen protein productive rate that makes, output, quality is not high, so that biomedical high, last with the collagen protein price, influenced the widespread use of collagen protein greatly, therefore, reducing collagen protein purifying cost is crucial with improving protein mass.

Summary of the invention:

The objective of the invention is to provide a kind of from the method for shark skin separation and purification biological medicine with collagen protein, it not only can extract the high quality collagen protein effectively from shark skin, and technology is simple, and with low cost, productive rate is higher.

To be achieved in that of the present invention be shark skin from shark skin separation and purification biological medicine with the employed raw material of the method for collagen protein in the present invention to achieve the above object, and therefrom the biomedical method operation with collagen protein of separation and purification is as follows:

(1), be that the low phosphorus phthalate buffer soaking flushing of 7.4-7.6 is removed squama and residual meat with shark skin with the pH value, and be cut into small pieces, use the deionized water soaking flushing again 3～5 times;

(2), the fish-skin that cleans up and be that homogenate 15min was 4 ℃ in temperature after the middle phosphorus concentration phthalate buffer of 7.4-7.6 soaked 70～75 hours with the pH value of five times of material quantities, rotating speed is 10000rpm, centrifugal 15min gets supernatant liquor;

(3), redeposition repeat (2) process once, will merge supernatant liquor, add ammonium sulfate to saturation ratio 80%, be 4 ℃ in temperature, rotating speed is 8000rpm, centrifugal 15min keeps and precipitates;

(4), precipitation being dissolved in the pH value is in 7.0 the middle phosphorus concentration acid buffer;

(5), use G-75, the above-mentioned lysate of 100,150 dextran chromatographies, collecting the albumen dissolution fluid is 4 ℃ in temperature, rotating speed is 10000rpm, centrifugal 15min, supernatant concentration obtains the biomedicine collagen protein after the lyophilize.

Described low phosphorus phthalate buffer is that concentration is 8-12mM, the pH value is the Sodium phosphate dibasic damping fluid of 7.4-7.6, the phosphorus concentration phthalate buffer is that concentration is 80-120mM in described, the pH value is the Sodium phosphate dibasic damping fluid of 7.4-7.6, the concentration of phosphorus concentration acid buffer is 180-220mM in described, the pH value is 7.0, and its consumption is 1.5g/ml.

Because the present invention is material with the shark skin, extract and collagen purification albumen by methods such as alkali cleaning, dialysis, therefore have the following advantages:

1, the shark skin abundant raw material is cheap, generally as tankage, as the protein extraction raw material, greatly reduces cost with it, it is reported, shark onal in the market, scalp price be at 6000-8000 unit/ton, about 1000 tons of gross annual output amounts;

2, to have reached electrophoresis after testing pure for the gained collagen protein, the quality height;

3, gained shark skin collagen protein can be dissolved in neutrality and the acidic solution, than the collagen protein in other sources bigger solvability is arranged, and can be applied to more areas;

4, gained shark skin collagen protein denaturation temperature is significantly higher than other source protein denaturation temperature, and this has unique application value on as biomedical material;

5, cost is low, and technical process is subjected to the restriction of instrument, material etc. little when amplifying, and only needs to increase human cost and gets final product.

Embodiment:

Below will be described in further detail from the method for shark skin separation and purification biological medicine of the present invention with collagen protein.

Used material is a horse glue shark skin in the present embodiment.

Concrete steps are as follows:

1, the horse glue shark skin 30g concentration with stored frozen is 10mM, and the pH value is removed squama and residual meat for the Sodium phosphate dibasic damping fluid soaking flushing of 7.4-7.6, is cut into 0.5cm * 0.5cm fritter, uses the deionized water soaking flushing again 3～5 times;

2, the fish-skin concentration that cleans up is 100mM, and the pH value is that the Sodium phosphate dibasic damping fluid 250ml of 7.4-7.6 soaked after 72 hours, and homogenate 15min is that 4 ℃, rotating speed are 10000rpm, centrifugal 15min with temperature, gets supernatant liquor;

3, the process of precipitation repetition 2 once merges supernatant liquor, adds ammonium sulfate to saturation ratio 80%, is 8000rpm with the rotating speed, and temperature is 4 ℃, and centrifugal 15min keeps precipitation;

4, the concentration that precipitation is dissolved in about 20ml is 200mM, and the pH value is in 7.0 the phosphoric acid buffer;

5, use G-75, the above-mentioned lysate of 100,150 dextran chromatographies, collecting the albumen dissolution fluid is that 4 ℃, rotating speed are 10000rpm in temperature, centrifugal 15min, supernatant concentration obtains the biomedicine collagen protein after the lyophilize.

Horse glue shark collagen through above-mentioned separation, purifying embodies three bands of a spectrum clearly on the SDS-PAGE of concentrated glue 4-5%, separation gel 7.5-8%, lower molecular weight peptide chain content is higher, and molecular mass is respectively 134kDa and 118kDa; High molecular peptide chain content is less, and its molecular mass is about 205kDa.This high molecular peptide chain is than the molecular weight height of the beta-peptide chain that obtains from land and other marine fishes.

The denaturation temperature of the collagen protein that is separated to from horse glue shark skin is 40.31 ℃, this exists beta sheet and random coil consistent with the space structure of collagen protein, this denaturation temperature significantly is different from the denaturation temperature of the marine fishes collagen protein of measuring with the viscosity method, from the thermal denaturation temperature of Japanese perch collagen protein is 30 ℃, 29.7 ℃ of the denaturation temperatures of tuna, the thermal denaturation temperature of fresh water fish scale is 32.3 ℃, the denaturation temperature of cow leather collagen is that 38 ℃ the fact is inferred, the collagen protein that is separated to from the blue shark scalp has unique spatial structure characteristic and amino acid formation characteristics, and this has the using value with uniqueness on as biomedical material.

Claims

1, a kind of from shark skin the biomedical method of separation and purification with collagen protein, it is characterized in that choosing raw material is shark skin, the preparation method is as follows:

(3), redeposition repeat (2) process once, merge supernatant liquor, add ammonium sulfate to saturation ratio 80%, be 4 ℃ in temperature, rotating speed is 8000rpm, centrifugal 15min keeps precipitation;

2, according to claim 1 from shark skin the biomedical method of separation and purification with collagen protein, it is characterized in that described low phosphorus phthalate buffer is that concentration is 8-12mM, the pH value is the Sodium phosphate dibasic damping fluid of 7.4-7.6.

3, according to claim 1 from shark skin the biomedical method of separation and purification with collagen protein, it is characterized in that described in the phosphorus concentration phthalate buffer be that concentration is 80-120mM, the pH value is the Sodium phosphate dibasic damping fluid of 7.4-7.6.

4, according to claim 1 from shark skin the biomedical method of separation and purification with collagen protein, it is characterized in that described in the concentration of phosphorus concentration acid buffer be 180-220mM, the pH value is 7.0, its consumption is 1.5g/ml.