1. An optimization method for extracting and purifying type I collagen from human placenta is characterized in that: the method comprises the following steps:

step (1), raw material pretreatment: unfreezing and cleaning the detected qualified human placenta, then shearing, draining and weighing for later use;

step (2), alkaline soaking: adding 0.5M NaOH solution into pretreated placenta tissue at W/V ratio of 1:10, stirring and soaking at 4 deg.C for 24 hr, and soaking at 1000 deg.C_╳g, centrifuging for 15min, collecting precipitate, adding 0.5M NaOH solution at W/V ratio of 1:5, stirring and soaking at 4 deg.C for 24 hr, 1000_╳g, centrifuging for 15min, and collecting precipitates;

step (3), acid soaking: adding 0.5M acetic acid solution into the collected precipitate according to the W/V ratio of 1:8, stirring and soaking at 4 deg.C for 12h, 1000_╳g, centrifuging for 15min, and collecting precipitates;

step (4), enzymolysis: adding 0.5M acetic acid or 0.5M citric acid solution into the precipitate at W/V1:5 ratio, adding pepsin at a ratio of 5mg pepsin per gram of tissue precipitate, adjusting pH to 2.4, stirring at 4 deg.C for enzymolysis for 24 hr, 14000_╳g, centrifuging for 15min, collecting the supernatant, storing in a refrigerator at 4 deg.C, and performing enzymolysis on the precipitate under the same conditionsSecondly, to fully extract collagen protein, 14000_╳g, centrifuging for 15min, removing precipitates, and collecting a centrifugal supernatant;

step (5), ultrafiltration concentration and buffer replacement: mixing the centrifugal supernatant, and concentrating by 2 times by using a 30kD polyether sulfone ultrafiltration membrane, so that the working volume is reduced, the protein concentration is improved, the subsequent process operation is facilitated, and the operation time is shortened; continuously adding pH6.80.1M PB buffer solution 5DV after concentration to wash, filter and replace buffer;

step (6), anion exchange chromatography;

step (7), ultrafiltration concentration and buffer replacement;

salting out;

step (9), dialysis: adding the salting-out precipitate into PBS buffer solution with pH value of 7.220mM according to the W/V ratio of 2:1, stirring into paste, and dialyzing the PBS buffer solution with pH value of 7.220mM at the temperature of 4 ℃ to keep the buffer solution in a physiological salt balance state, thereby obtaining the high-purity type I collagen.

2. The optimized method of claim 1 for extracting purified type I collagen from human placenta, wherein the method comprises the steps of: the step (1) comprises removing surface bloodiness, cutting off umbilical cord, adding 9% NaCl + 3% ethanol solution according to W/V ratio of 1:4, and cleaning for 5 times to fully remove soluble blood components, lipid and foreign protein.

3. The optimized method of claim 1 for extracting purified type I collagen from human placenta, wherein the method comprises the steps of: the anion exchange chromatography in the step (6) refers to that the concentrated and replaced buffer liquid in the previous step is put on a DEAE chromatographic column at the temperature of 4 ℃; the chromatographic conditions are as follows: 1) the balance buffer solution is pH6.80.1MPB; 2) RT is 20 h; collecting the flow-through peak feed liquid.

4. The optimized method of claim 1 for extracting purified type I collagen from human placenta, wherein the method comprises the steps of: and (3) concentrating and replacing the buffer in the step (7) refers to concentrating the DEAE chromatography flow-through peak feed liquid by 8 times by using a 30kD polyether sulfone ultrafiltration membrane so that the feed liquid is in the optimal salting-out concentration, and continuously supplementing a 0.5M citric acid solution 5DV after the concentration is finished, washing, filtering and replacing the buffer.

5. The optimized method of claim 1 for extracting purified type I collagen from human placenta, wherein the method comprises the steps of: the salting-out in the step (8) is to slowly add solid NaCl into the feed liquid while stirring to completely dissolve the solid NaCl until the final concentration is 2M, standing for 12h at 4 ℃, and 14000_╳g, centrifuging for 15min, and separating out type I collagen; discarding supernatant, adding 0.5M citric acid solution into the precipitate according to W/V ratio of 1:10 for redissolving, 14000_╳g, centrifuging for 15min, and removing precipitates to remove insoluble collagen; slowly adding solid NaCl into the supernatant while stirring to completely dissolve NaCl to final concentration of 2M, standing at 4 deg.C for 12 hr, 14000%_╳g, centrifuging for 15min, further purifying and separating out the type I collagen, and collecting the precipitate.

Background

Human placenta, as a temporary organ for maintaining the growth and development of fetus in mother, has been regarded as important since ancient times because of its special barrier function and endocrine function. In recent years, the research on the placenta is gradually and deeply carried out, and the process is also developed from simple drying and water boiling to biological extraction, so that people can more and more deeply understand the beneficial components and bioactive substances contained in the placenta. Among them, extraction of collagen from human placenta is one of the research directions of interest. Collagen is a biopolymer synthesized by animal fibroblasts, is a white, opaque, unbranched fibrous protein, and is mainly present in animal tendons, ligaments, cartilage, skin, and other connective tissues. The human placenta is distributed with a great amount of connective tissues, has rich collagen content and is a good material for extracting the collagen.

Collagen has the advantages of biodegradability, low immunogenicity, feasibility of large-scale production and the like due to the special structure and special performance of the collagen, is an important natural polymer biomaterial, and has increasingly prominent application and economic significance in the industries of food, biomedicine, pharmacology, cosmetics and the like. At present, the research on collagen mainly focuses on the theoretical and practical aspects, such as the research on the manufacturing method, the physicochemical property, the biological property and the like of materials, so that a plurality of valuable achievements are obtained, and the industrial application is common. Among the members of the collagen family, type I collagen is the most well-understood collagen and the most widely distributed collagen in tissues, and has a very high application value.

At present, the collagen is mainly derived from animal skin, horn, bone, tendon, cartilage tissue and other heterologous materials, possibly has pathogenic pollution and high immunogenicity, and possibly stimulates a certain immune response. The human placenta has homology, low immunoreaction and very high application value. However, the method has the problems of low purity, long production period, low yield, difficult industrial preparation and the like of collagen extracted and prepared from human placenta. Therefore, the increasing demand for collagen is not satisfied, either quantitatively or qualitatively. At present, the technology for producing proteins for medicines, foods and cosmetics by applying biotechnology is mature day by day, so the production and development of humanized collagen have good application prospect.

Disclosure of Invention

The technical problem to be solved by the present invention is to provide an optimized method for extracting and purifying type I collagen from human placenta, so as to solve the problems proposed in the background art.

The technical problem solved by the invention is realized by adopting the following technical scheme: an optimization method for extracting and purifying type I collagen from human placenta comprises the following steps:

step (1), raw material pretreatment: unfreezing and cleaning the detected qualified human placenta, then shearing, draining and weighing for later use;

step (2), alkaline soaking: adding 0.5MNaOH solution into pretreated placenta tissue according to a W/V ratio of 1:10, stirring and soaking for 24 hours at 4 ℃, centrifuging for 15 minutes at 1000 x g, collecting precipitates, adding 0.5MNaOH solution according to a W/V ratio of 1:5, stirring and soaking for 24 hours at 4 ℃, fully swelling the tissue, removing lipid and inactivating viruses, centrifuging for 15 minutes at 1000 x g, and collecting the precipitates;

step (3), acid soaking: adding 0.5M acetic acid solution into the collected precipitate according to the W/V ratio of 1:8, stirring and soaking at 4 ℃ for 12h to passivate the antigen and remove soluble components, centrifuging at 1000 x Xg for 15min, and collecting the precipitate;

step (4), enzymolysis: adding 0.5M acetic acid or 0.5M citric acid solution into the precipitate according to a W/V1:5 ratio, adding 5mg pepsin into each gram of tissue precipitate, correcting the pH value to 2.4, stirring for enzymolysis for 24 hours at 4 ℃, centrifuging for 15min at 14000 mmg, collecting centrifugal supernatant, temporarily storing the centrifugal supernatant in a refrigerator at 4 ℃, performing enzymolysis extraction once again on the precipitate under the same condition to fully extract collagen, centrifuging for 15min at 14000 mmg, removing the precipitate, collecting centrifugal supernatant, increasing the enzymolysis frequency and improving the yield;

step (5), ultrafiltration concentration and buffer replacement: mixing the centrifugal supernatant, and concentrating by 2 times by using a 30kD polyether sulfone ultrafiltration membrane, so that the working volume is reduced, the protein concentration is improved, the subsequent process operation is facilitated, and the operation time is shortened; after the concentration is finished, the pH6.80.1MPB buffer solution 5DV is continuously supplemented, the filter washing and the buffer replacement are carried out, so that the balance condition of the buffer solution is the same as that of the subsequent anion chromatography, most of small molecular impurities are removed, the preliminary separation and purification effect is achieved, the ultrafiltration replacement buffer replaces dialysis, and the operation time can be obviously shortened;

step (6), anion exchange chromatography;

step (7), ultrafiltration concentration and buffer replacement;

salting out;

step (9), dialysis: adding the salting-out precipitate into pH7.220mMPBS buffer solution according to the W/V ratio of 2:1, stirring into paste, and dialyzing the pH7.220mMPBS buffer solution at 4 ℃ to keep the buffer solution in a physiological salt balance state to obtain the high-purity type I collagen.

The step (1) comprises removing surface bloodiness, cutting off umbilical cord, adding 9% NaCl + 3% ethanol solution according to W/V ratio of 1:4, and cleaning for 5 times to fully remove soluble blood components, lipid and foreign protein.

And (3) anion exchange chromatography in the step (6) is to perform DEAE chromatography on the concentrated and replaced buffer solution in the last step at the temperature of 4 ℃. So as to remove harmful substances such as pepsin, endotoxin, acidic macromolecules and the like, ensure the safety of the product and have the pure effect. The chromatographic conditions are as follows: 1) the balance buffer solution is pH6.80.1MPB;

2) RT was 20 h. Collecting the flow-through peak feed liquid.

And (3) concentrating and replacing the buffer in the step (7) means that DEAE chromatography flow-through peak feed liquid is concentrated by 8 times by using a 30kD polyether sulfone ultrafiltration membrane, so that the feed liquid is in the optimal salting-out concentration, 0.5M citric acid solution 5DV is continuously supplemented after the concentration is finished, the washing and filtering are performed, the buffer is replaced, so that the buffer solution and impurities are removed, and meanwhile, the feed liquid is in an acidic condition, so that the type I collagen is easily separated by salting-out.

And (3) performing salting-out in the step (8) by slowly adding solid NaCl into the feed liquid while stirring to completely dissolve the solid NaCl until the final concentration is 2M, standing for 12h at 4 ℃, centrifuging at 14000 x g for 15min, and separating out the type I collagen. Discarding supernatant, adding 0.5M citric acid solution into the precipitate according to W/V ratio of 1:10 for redissolving, centrifuging at 14000 x g for 15min, and discarding the precipitate to remove insoluble collagen. Slowly adding solid NaCl into the supernatant while stirring to completely dissolve NaCl to a final concentration of 2M, standing at 4 ℃ for 12h, centrifuging at 14000 gamma g for 15min, further purifying and separating out type I collagen, and collecting the precipitate.

Compared with the prior art, the invention has the beneficial effects that: the method avoids the defects caused by singly using the acid soaking process or the alkali soaking process by simultaneously using the alkali soaking process and the acid soaking process, increases the times of enzymolysis, fully extracts the target protein, increases the extraction amount of the target protein, increases the removal amount of impurities, can obviously improve the yield of the target protein, and can obviously reduce the preparation time and shorten the production period by replacing the traditional dialysis process with ultrafiltration concentration and buffer replacement.

In conclusion, the method can obviously improve the productivity, is easy to enlarge the production, greatly shortens the preparation time, improves the purity, is easy to form products and has wider application field.

The I type collagen (type I collagen) peptide chain is composed of repeated Gly-X-Y amino acid sequences, all collagens have a triple-helix structure composed of three polypeptide chain units and are formed by alternately winding 2 alpha 1 chains and 1 alpha 2 chain. The α 2 chain is a characteristic chain of type I collagen, so the presence of the α 2 chain indicates the presence of type I collagen. The type I collagen is rich in glycine (Gly), L-alanine (L-Ala), L-proline (L-Pro) and 4-hydroxyproline (4-Hypro), has low sulfur content, and does not contain L-tryptophan (L-Try).

Other proteins other than type I collagen remaining during collagen purification include (but are not limited to): other structural molecules of elastin (amino polysaccharides, lipoprotein and glycoprotein complexes, etc.), incorrectly aligned collagen molecules, enzyme preparations, and the like. Type iii collagen, which may accompany type I collagen, is not considered a hetero protein.

Drawings

FIG. 1 is a flow chart of the preparation process of the present invention.

FIG. 2 is an SDS-PAGE pattern of human placental type I collagen according to the invention.

Detailed Description

In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention is further described below by combining the specific drawings.

Human placenta source

The raw material human placenta is a healthy human placenta which is collected by strict aseptic operation and is used for producing biological products, and in order to ensure the quality and the safety of the human placenta, the collected contents comprise the blood plasma of a puerpera, the blood plasma of the placenta, an informed contract signed by the puerpera and a complete placenta blood and placenta collection registration form. The collection hospital is more than second class A and the like, and has the primary screening detection qualification of HBV, HCV, TP and HIV.

According to the requirements of 'Chinese pharmacopoeia' 2020 edition and 'quality control standard of main raw and auxiliary materials of Chinese biological products', the collected material placenta is strictly screened, and the collected placenta is subjected to HBsAg, syphilis, HIV-1/HIV-2 antibody and HCV antibody examination by adopting a kit approved by the national drug administration, and the placenta prescription of the total negative person can be put into use.

Second, preparation method

As shown in fig. 1, an optimized method for extracting and purifying type I collagen from human placenta comprises the following steps:

step (1), raw material pretreatment: thawing the detected qualified human placenta, removing surface blood stains, shearing umbilical cord, adding 9% NaCl + 3% ethanol solution according to the W/V ratio of 1:4, cleaning for 5 times to fully remove soluble blood components, lipid and foreign proteins, shearing, filtering, and weighing for later use;

step (2), alkaline soaking: adding 0.5MNaOH solution into pretreated placenta tissue according to a W/V ratio of 1:10, stirring and soaking for 24 hours at 4 ℃, centrifuging for 15 minutes at 1000 x g, collecting precipitates, adding 0.5MNaOH solution according to a W/V ratio of 1:5, stirring and soaking for 24 hours at 4 ℃, fully swelling the tissue, removing lipid and inactivating viruses, centrifuging for 15 minutes at 1000 x g, and collecting the precipitates;

step (3), acid soaking: adding 0.5M acetic acid solution into the collected precipitate according to the W/V ratio of 1:8, stirring and soaking at 4 ℃ for 12h to passivate the antigen and remove soluble components, centrifuging at 1000 x Xg for 15min, and collecting the precipitate;

step (4), enzymolysis: adding 0.5M acetic acid or 0.5M citric acid solution into the precipitate according to a W/V1:5 ratio, adding 5mg pepsin into each gram of tissue precipitate, correcting the pH value to 2.4, stirring for enzymolysis for 24 hours at 4 ℃, centrifuging for 15min at 14000 mmg, collecting centrifugal supernatant, temporarily storing in a refrigerator at 4 ℃, performing enzymolysis extraction once again on the precipitate under the same condition to fully extract collagen, centrifuging for 15min at 14000 mmg, removing the precipitate, and collecting the centrifugal supernatant;

step (5), ultrafiltration concentration and buffer replacement: mixing the centrifugal supernatant, concentrating by 2 times by using a 30kD polyether sulfone ultrafiltration membrane, reducing the working volume, improving the protein concentration, facilitating the subsequent process operation and shortening the operation time; after the concentration is finished, the pH6.80.1MPB buffer solution 5DV is continuously supplemented, the filter washing and the buffer replacement are carried out, so that the buffer solution has the same balance condition with the subsequent anion chromatography, and most of small molecular impurities are removed, thereby having the primary separation and purification effect;

step (6), anion exchange chromatography: collecting peak-crossing feed liquid, and removing harmful substances such as pepsin, endotoxin, acidic macromolecules and the like to ensure the safety of the product and have a fine and pure effect; concentrating and replacing buffer liquid, and loading on a DEAE chromatographic column at 4 ℃, wherein the chromatographic conditions are as follows: the balance buffer solution is pH6.80.1MPB; RT is 20 h;

step (7), ultrafiltration concentration and buffer replacement: concentrating the DEAE chromatography flow-through peak feed liquid by using a 30kD polyether sulfone ultrafiltration membrane for 8 times to ensure that the feed liquid is in the optimal salting-out concentration, continuously supplementing 0.5M citric acid solution 5DV after the concentration is finished, washing, filtering and replacing buffer to remove buffer solution and impurities, and simultaneously ensuring that the feed liquid is in an acidic condition to be easy to salt out and separate out type I collagen;

step (8), salting out: slowly adding NaCl solid while stirring to completely dissolve the NaCl solid to a final concentration of 2M, standing for 12h at 4 ℃, centrifuging for 15min at 14000 gamma g, and separating out type I collagen; discarding the supernatant, adding 0.5M citric acid solution into the precipitate according to the W/V ratio of 1:10 for redissolving, centrifuging at 14000 x gamma g for 15min, discarding the precipitate to remove insoluble collagen; slowly adding NaCl solid into the supernatant while stirring to completely dissolve the NaCl solid to a final concentration of 2M, standing at 4 ℃ for 12h, centrifuging at 14000 gamma g for 15min, further purifying and separating out type I collagen, and collecting precipitate;

step (9), dialysis: adding the salting-out precipitate into pH7.220mMPBS buffer solution according to the W/V ratio of 2:1, stirring into paste, and dialyzing the pH7.220mMPBS buffer solution at 4 ℃ to keep the buffer solution in a physiological salt balance state to obtain the high-purity type I collagen.

Third, experimental results

1. The appearance is milky viscous semisolid.

2. The molecular weight distribution of the type I collagen was measured by SDS-PAGE gel electrophoresis method according to the fifth method of 0541 in the pharmacopoeia of the people's republic of China (2015), and the results were consistent with the electrophoresis results of the type I collagen standards (see FIG. 2).

3. The content of the obtained I-type collagen is measured according to an appendix A of YY0954-2015 Passive surgical implant I-type collagen implant, namely a sirius red dyeing method, and the yield of the I-type collagen extracted and purified by the optimization method is obviously improved compared with the yield of the I-type collagen extracted and purified by the prior reported method (see table 1).

TABLE 1 type I collagen yield

Method	Placenta mass (g)	Collagen quality (g)	Yield (%)
				Existing methods	1000	1.02	0.102
The method of the invention	922	3.573	0.388

4. Checking tryptophan: according to the method specified in appendix E of YY0954-2015 Passive surgical implant type I collagen implant, a purplish red ring is not generated on the interface of acetic acid and sulfuric acid.

5. And (3) analysis of hybrid protein: the total amount of foreign proteins was below 1% of the total protein, as determined in appendix B of YY0954-2015 Passive surgical implant type I collagen implant.

6. The preparation period is as follows: compared with the traditional process, the method for extracting and purifying the type I collagen obviously shortens the operation time (see table 2).

TABLE 2 type I collagen preparation cycle

Method	Preparation period (Tian)
		Existing methods	15
The method of the invention	10

In conclusion, the human placenta-derived type I collagen prepared by the method has the advantages of improving the yield by more than 3 times, shortening the preparation periodThe quality is stable; the method is simple and feasible, is suitable for large-scale production, and has good application prospect.

The principal features of the invention and advantages of the invention have been shown and described above. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.