阅读说明：本技术 一种胶原的提取方法 (Collagen extraction method ) 是由 岳崇霞 宋磊 于 2019-09-25 设计创作，主要内容包括：本发明公开一种胶原的提取方法,包括：提供经预处理后的动物胎皮,得到预制件；将所述预制件经过脂肪处理剂浸泡处理,并用梯度浓度的酒精进行清洗,进行脱脂处理；将所述脱脂处理后的预制件,在酶消化液中进行消化处理；将所述消化处理后的混合物进行过滤、溶解、盐析、透析,得到胶原。本发明提供的方法在使用动物胎皮进行胶原提取时,能够提高胶原的收率,易操作,成本低。(The invention discloses a method for extracting collagen, which comprises the following steps: providing the pretreated animal fetal skin to obtain a prefabricated part; soaking the prefabricated member in a fat treating agent, cleaning the prefabricated member by using alcohol with gradient concentration, and performing degreasing treatment; digesting the degreased prefabricated member in an enzyme digestion solution; and filtering, dissolving, salting out and dialyzing the mixture after the digestion treatment to obtain the collagen. The method provided by the invention can improve the yield of collagen when the animal fetal skin is used for extracting the collagen, and is easy to operate and low in cost.)

1. A method for extracting collagen, comprising:

Providing the pretreated animal fetal skin to obtain a prefabricated part;

Soaking the prefabricated member in a fat treating agent, cleaning the prefabricated member by using alcohol with gradient concentration, and performing degreasing treatment;

Digesting the degreased prefabricated member in an enzyme digestion solution;

And filtering, dissolving, salting out and dialyzing the mixture after the digestion treatment to obtain the collagen.

2. The method according to claim 1, characterized in that the pre-treatment is in particular:

After 1-2 times of salt washing is carried out on the animal fetal skin, the residual salt water is removed;

Alkali washing the salted animal fetal skin in an alkaline solution for 1-8 h, and washing to be neutral;

Repeatedly freezing and unfreezing the animal fetal skin after alkali washing, and removing epidermis and subcutaneous fat;

carrying out buffer cleaning on the animal fetal skin with the epidermis and the subcutaneous fat removed in a buffer solution, and then washing to be neutral;

And repeatedly freezing and unfreezing the animal fetal skin after buffer cleaning to obtain the prefabricated member.

3. The method of claim 2, wherein the pretreatment is performed such that the ratio of the bulk weight of the feedstock to the total pretreatment fluid is an excess of pretreatment fluid.

4. The method according to claim 1, wherein the degreasing treatment is in particular:

Soaking the prefabricated member in lipase and/or organic solvent, soaking in gradient alcohol, and cleaning.

5. the method according to claim 4, wherein the volume ratio of the preform to lipase or organic solvent is 1: 5.

6. The method according to claim 4, wherein the gradient alcohol soaking is carried out by soaking in 98% alcohol for 4-8 h, 60-70% alcohol for 2-6 h, and 20-40% alcohol for 0.5-2 h.

7. The method of claim 4, wherein the organic solvent is selected from ethanol, chloroform, diethyl ether, or acetone.

8. The method according to claim 1, characterized in that the digestion treatment is in particular:

Soaking the degreased prefabricated member in an enzyme digestion solution in an ultrasonic environment until the digestion is complete; the weight ratio of the degreased prefabricated member to the enzyme digestion solution is 1: 5-15.

9. the method of claim 8, wherein the enzymatic digest is selected from the group consisting of an acidic solution of pepsin having a pH of 2.0-3.0.

10. The method according to claim 1, wherein the mixture after the digestion treatment is filtered with a 50-200 mesh sieve to remove undigested tissue and impurities;

dissolving in salt solution completely, and salting out for 15-30 hr;

Discharging the supernatant, adding the precipitated collagen into dialysis bag (10-80KD), dialyzing for 5-10 days, and changing the solution 2-3 times per day to obtain purified collagen.

Technical Field

the invention relates to the technical field of biology, in particular to a collagen extraction method.

background

Collagen is a high molecular protein synthesized by animal cells and has a triple-strand supercoiled structure, has good biological properties, and is a main structural protein of connective tissues. In the dermis of animals such as cattle, pigs and sheep, about 70% is collagen.

the collagen in the animal dermis can be used as a tissue support after being extracted and purified, can perform normal functions on cells, tissues and even organs, and has good auxiliary effect on the repair of trauma. Compared with the collagen extracted from the animal dermis of other age groups, the fetal skin (comprising fetal calf skin, fetal pig skin, fetal sheep skin and the like) has the advantages of stable collagen, easy extraction, higher collagen content and the like, so the fetal skin is more suitable for the biomedical material fields of cosmetic filling, cell scaffolds, orthopedics, tissue injury repair, wound hemostasis and the like.

Although the extraction of collagen from fetal skin is easier than the pretreatment of adult animal fur, inadequate collagen extraction and loss of collagen can occur in other steps if the control is not proper, and the fetal skin is relatively costly and the overall loss is relatively large if the collagen extraction is inadequate.

disclosure of Invention

In view of the above, the present invention provides a method for extracting collagen, which can improve the yield of collagen when using animal fetal skin for collagen extraction, and is easy to operate and low in cost.

The invention mainly realizes the purposes by the technologies of raw material pretreatment, degreasing, digestion, purification and the like, and comprises the following detailed steps:

providing pretreated animal fetal skin;

Soaking the prefabricated member in a fat treating agent, cleaning the prefabricated member by using alcohol with gradient concentration, and performing degreasing treatment;

Digesting the degreased prefabricated member in an enzyme digestion solution;

And filtering, dissolving, salting out and dialyzing the mixture after the digestion treatment to obtain the collagen.

Preferably, the pretreatment specifically comprises:

After 1-2 times of salt washing is carried out on the animal fetal skin, the residual salt water is removed;

Alkali washing the salted animal fetal skin in an alkaline solution for 1-8 h, and washing to be neutral;

repeatedly freezing and unfreezing the animal fetal skin after alkali washing, and removing epidermis and subcutaneous fat;

Carrying out buffer cleaning on the animal fetal skin with the epidermis and the subcutaneous fat removed in a buffer solution, and then washing to be neutral;

And repeatedly freezing and unfreezing the animal fetal skin after buffer cleaning to obtain the prefabricated member.

preferably, the weight ratio of the raw material to the total weight of the pretreatment liquid in the pretreatment is excessive.

Preferably, the degreasing treatment specifically comprises:

soaking the prefabricated member in lipase and/or organic solvent, soaking in gradient alcohol, and cleaning after soaking;

Preferably, the volume ratio of the preform to the lipase or organic solvent is 1: 5.

preferably, the gradient alcohol soaking is to soak in 98% alcohol by volume for 4-8 h, 60-70% alcohol by volume for 2-6 h, and 20-40% alcohol by volume for 0.5-2 h.

Preferably, the organic solvent is selected from ethanol, chloroform, diethyl ether, or acetone.

Preferably, the digestion treatment specifically comprises:

soaking the degreased prefabricated member in an enzyme digestion solution in an ultrasonic environment until the digestion is complete; the weight ratio of the degreased prefabricated member to the enzyme digestion solution is 1: 5-15.

Preferably, the enzymatic digest is selected from an acidic solution of pepsin having a pH of 2.0-3.0.

Preferably, the mixture after the digestion treatment is filtered by a 50-200 mesh filter screen to remove undigested tissues and impurities;

adding salt until the salt is completely dissolved, and salting out for 15-30 hours;

discharging the supernatant, adding the precipitated collagen into dialysis bag (10-80KD), dialyzing for 5-10 days, and changing the solution 2-3 times per day to obtain purified collagen.

The invention provides a collagen extraction method, which comprises the steps of providing pretreated animal fetal skin to obtain a prefabricated part; soaking the prefabricated member in a fat treating agent, cleaning the prefabricated member by using alcohol with gradient concentration, and performing degreasing treatment; digesting the degreased prefabricated member in an enzyme digestion solution; and filtering, dissolving, salting out and dialyzing the mixture after the digestion treatment to obtain the collagen. The invention can provide relatively stable collagen with high purity by using the fetal skin with unformed hair as a raw material. The method of repeatedly swelling and freezing and thawing fetal skin by using alkali and high-concentration salt can obviously divide the fetal skin into three layers (epidermis, dermis and subcutaneous fat), can quickly remove the epidermis and the subcutaneous fat by using a scraping rod and reserve the dermis for extracting collagen, and meanwhile, the method can also effectively remove non-collagen impurity protein and improve the purity and the yield of the collagen by increasing a fat treatment agent and an enzyme digestive juice. The appropriate ultrasonic time and power are selected to assist the enzyme method, so that the extraction efficiency of the collagen can be effectively improved, the digestion and extraction time of the collagen can be saved, and the obtained collagen can maintain the natural structure to the maximum extent and is suitable for being used as biomedical materials.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present invention, the present invention will be further described in detail with reference to the following embodiments.

In order to further explain the technical scheme of the invention in detail, the specific collagen extraction steps are as follows:

The yield mentioned in the embodiments of the present invention refers to the ratio of the weight of collagen to the weight of the raw material.

in order to solve the technical problems of the invention, the invention provides a method for extracting collagen,

providing the pretreated animal fetal skin to obtain a prefabricated part; soaking the prefabricated member in a fat treating agent, cleaning the prefabricated member by using alcohol with gradient concentration, and performing degreasing treatment; digesting the degreased prefabricated member in an enzyme digestion solution; and filtering, dissolving, salting out and dialyzing the mixture after the digestion treatment to obtain the collagen.

The pretreatment is a step of cleaning impurities on fur, washing hair and separating a fat layer, a dermis layer and collagen, and is a prerequisite condition for providing a good base for the subsequent steps of degreasing, digestion and purification, and because the invention provides animal fetal skin, the hair is soft, the dermis layer and fat are less, the hair is relatively immature, and the condition requirements are not strict in the pretreatment process, but the pretreatment is carried out carefully to prevent the collagen of the animal fetal skin from being damaged.

the pretreatment steps of the invention are specifically as follows: after 1-2 times of salt washing is carried out on the animal fetal skin, the residual salt water is removed; alkali washing the salted animal fetal skin in an alkaline solution for 1-8 h, and washing to be neutral; repeatedly freezing and unfreezing the animal fetal skin after alkali washing, and removing epidermis and subcutaneous fat; carrying out buffer cleaning on the animal fetal skin with the epidermis and the subcutaneous fat removed in a buffer solution, and then washing to be neutral; and repeatedly freezing and unfreezing the animal fetal skin after buffer cleaning to obtain the prefabricated member.

Wherein, the salt washing preferably uses sodium chloride (NaCl) water solution, can effectively clean impurities on the surface of the fetal skin, and has certain swelling effect to dehydrate the animal fetal skin. The concentration of NaCl is preferably 0.2-0.5M/L, and more preferably 0.3-0.4M/L. The washing time is preferably 1-5 h, more preferably 2-4 h, the salt washing is preferably carried out once, the cleaning solution is poured off, a new sodium chloride aqueous solution is replaced, the cleaning is repeated, and the stirring is continuously carried out in the cleaning process.

After the animal fetal skin is salted, removing a sodium chloride aqueous solution remained on the surface of the animal fetal skin by using deionized water, and carrying out alkali washing in an alkaline solution, wherein the alkaline aqueous solution is preferably sodium hydroxide NaOH, and the concentration of the sodium hydroxide aqueous solution is 0.05-0.5M/L, more preferably 0.08-0.2M/L. The alkali washing time is preferably 1-8 h, more preferably 2-6 h, after the alkali washing is completed once, the alkali washing is repeated for 2 times by replacing a new sodium hydroxide aqueous solution, and stirring is continuously performed in the washing process. After the alkali washing is finished, deionized water is used for washing until the solution is neutral.

after the alkaline washing is finished, preferably, NaCl-Tris-HCl aqueous solution is used for washing the animal fetal skin, the concentration of the NaCl-Tris-HCl aqueous solution is preferably 1-5M/L, the washing time is preferably 1-3 h, preferably, the washing is repeated for 1-2 times, and then, deionized water is used for washing until the animal fetal skin is neutral. The purpose of the buffer solution is here to remove the remaining cell tissue even further.

After the first buffer solution washing, the skin is frozen and stored for 10 to 18 hours at the temperature of between 90 ℃ below zero and 70 ℃ below zero, then the skin is taken out for thawing, the thawing is repeated for 2 times, the skin and subcutaneous fat are scraped after the last thawing, and the collagen cannot be damaged, so that the skin and subcutaneous fat are scraped carefully.

and after the final thawing, adding NaCl-Tris-HCl aqueous solution again for carrying out secondary buffer cleaning, wherein the concentration of the NaCl-Tris-HCl aqueous solution is preferably 1-5M/L, the cleaning time is preferably 1-3 h, washing is preferably repeated for 1-2 times, and then the solution is cleaned to be neutral by deionized water. The purpose of the buffer solution is here to remove the remaining cell tissue even further.

Preferably, after the second buffer washing, the animal fetal skin is laid on a 2cm tray, frozen at-80 ℃ for 10-18 hours, taken out for thawing, and cut into about 1cm by a cutter when the animal fetal skin is not completely thawed²and (5) blocking to obtain a prefabricated member.

According to the invention, the weight of the animal hides in said pretreatment may be excessive compared to the total weight of all the pretreatment liquids, but in order to avoid waste, it is more preferable to select 6 times the weight of the total weight of the pretreatment liquids, including sodium chloride, sodium hydroxide and NaCl-Tris-HCl solution, as well as clear water.

soaking the prefabricated member in lipase and/or organic solvent, soaking in gradient alcohol, and cleaning after soaking; the volume ratio of the prefabricated member to the lipase or the organic solvent is 1: 5. the gradient alcohol soaking is specifically to soak in 98% alcohol by volume for 4-8 h, 60-70% alcohol by volume for 2-6 h, and 20-40% alcohol by volume for 0.5-2 h.

the organic solvent is selected from ethanol, chloroform, diethyl ether, or acetone.

The digestion treatment specifically comprises the following steps: soaking the degreased prefabricated member in an enzyme digestion solution in an ultrasonic environment until the digestion is complete; the weight ratio of the degreased prefabricated member to the enzyme digestion solution is 1: 5-15. The enzyme digest is selected from acidic solutions of pepsin having a pH of 2.0-3.0. .

The enzyme digestive juice is dissolved in solution of acetic acid, citric acid, hydrochloric acid, etc. with pH of 2.0-3.0, and the specific gravity of the material and pepsin is 100: 1-7.

The ultrasonic wave is extracted by ultrasonic wave with ultrasonic time of 20-60min and ultrasonic power of 50-300W.

Filtering the mixture after digestion treatment by using a 50-200 mesh filter screen to remove undigested tissues and impurities; adding salt until the salt is completely dissolved, wherein the salt is NaCl, the final concentration is 1M/L, and salting out for 15-30 hours; discharging the supernatant, adding 2 times of hydrochloric acid solution with the volume of 0.01M/L for ultrasonic-assisted dissolution for 5-10 hours, adding ground NaCl until the concentration is 2.5M/L, fully stirring until the NaCl is completely dissolved, and salting out for 15-30 hours; removing the supernatant, adding the precipitate into a dialysis bag (10-80KD), dialyzing for 5-10 days, changing the solution for 1 time every 8-10 hours, and then performing low-temperature aseptic storage.

The ultrasonic-assisted dissolution: and (3) performing ultrasonic assisted extraction by using 20KHz ultrasonic waves for 30min at an ultrasonic power of 200W to obtain the collagen with the maximum extraction rate.

The above operation processes are all aseptic operation without special description and are carried out at 4 ℃.

The following are specific embodiments of the present invention: