CN113201569A

CN113201569A - Purification method of bovine type I collagen

Info

Publication number: CN113201569A
Application number: CN202110685663.1A
Authority: CN
Inventors: 陈敬华; 许淑琴; 毛宝亮
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-06-21
Filing date: 2021-06-21
Publication date: 2021-08-03
Anticipated expiration: 2041-06-21
Also published as: CN113201569B

Abstract

The invention relates to a method for purifying cattle type I collagen, which comprises the following steps: dissolving the bovine type I collagen subjected to crude extraction treatment in an acid solution, degrading by using a proteolytic enzyme of non-collagenase, and monitoring viscosity to control the enzymolysis degree; adjusting the pH value of the supernatant to 3.5-5, sequentially dropwise adding alcohol solutions with final concentrations of 15-25% v/v, 30-45% v/v, 50-70% v/v and 75-95% v/v to separate out 4 telopeptide bovine type I collagen, centrifuging, redissolving in an acid solution, adjusting to an isoelectric point, centrifuging to obtain a precipitate, redissolving in an acid solution, dialyzing in an acid solution with the pH value of 3-5, filtering for sterilization, taking a filtrate, and freeze-drying to obtain the purified bovine type I collagen. The bovine type I collagen provided by the invention has high purity, no immunogenicity and a sterile active form, and can be applied to the field of medical and tissue engineering materials.

Description

Purification method of bovine type I collagen

Technical Field

The invention relates to the field of protein purification, in particular to a purification method of bovine type I collagen.

Background

Collagen is the most abundant protein in many vertebrates and invertebrates, accounts for 25% -33% of the total protein mass, widely exists in skin, tendon and bone of animals, and the extracted products thereof are widely applied to a plurality of fields such as medicine, health care, food processing, cosmetics and the like. To date, 29 types of collagen have been found, and more studied are type i-v collagen, which is the predominant protein in bone, skin, tendon and other fibrous connective tissues, accounting for 90% of the total collagen in the organism. The type I collagen is fibrillogenic collagen, can activate epithelial cells, promote the proliferation of cells of raw skins, can also promote the generation of collagenase, has tension and elasticity on skins, and is used for repairing skin wounds and bone structure injuries in the field of medicines.

The N-and C-termini of type i collagen molecules have non-helical domains called telopeptides, which determine the antigenicity of the collagen, reducing its biocompatibility. In the prior art, crude extraction is usually carried out at the early stage of protein extraction and purification, and then collagen is purified through precipitation, centrifugation and the like, but the method is difficult to obtain high-purity type I collagen.

Patent CN1304416A discloses a method for preparing native collagen in a liquid medium, in which a mixture with a double-layer transverse cutter is stirred, a collagen extract is sheared, and then sterilized to obtain sterile collagen. However, the redundant mechanical shearing, while reducing the size of the collagen, can also lead to incomplete helical structure of the collagen; patent CN106946988A discloses a method for extracting bovine achilles tendon collagen, which separates and purifies type I collagen by a method combining enzymolysis and high performance liquid chromatography preparation; liulili and the like (bovine bone I type collagen extraction and structure characterization, food science, 2010, 31(02):87-91) adopt ether for low-temperature reflux degreasing, 0.48 percent of hydrochloric acid is decalcified, the bone particle size is 5mm multiplied by 10mm, and 1 percent of citric acid and 1 percent of pepsin are compounded as an extraction medium to extract the bovine bone I type collagen; wen Liu Wen et al (optimizing the purification process of type I collagen, fine chemical engineering, 2019, 36(05): 850-. However, there are problems of a small extraction amount or a low degree of purification. Therefore, there is still a need for a method for purifying bovine type I collagen with high extraction efficiency and high purity.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for purifying cattle type I collagen, and the cattle type I collagen has high purity, no immunogenicity and a sterile active form.

The invention relates to a cattle type I collagen purification method, which comprises the following steps:

(1) dissolving the roughly extracted bovine type I collagen, mixing and stirring the bovine type I collagen with a proteolytic enzyme of non-collagenase, monitoring the viscosity change, centrifuging after completing enzymolysis, and taking supernatant, wherein the viscosity of reaction liquid reaches 0.01-1 Pa.S to indicate that the enzymolysis is completed;

(2) adjusting the pH value of the supernatant in the step (1) to 3.5-5, sequentially dropwise adding alcohol solutions with final concentrations of 15-25% v/v, 30-45% v/v, 50-70% v/v and 75-95% v/v, sequentially separating out 4 collagen precipitates, analyzing the de-telopeptide degree by SDS-PAGE, and combining to obtain a de-telopeptide bovine I type collagen precipitate;

(3) and (3) redissolving the telopeptide bovine I type collagen sediment obtained in the step (2) in an acid solution with the pH value of 2-4, adjusting to an isoelectric point, centrifuging to obtain the sediment, redissolving in an acid solution with the pH value of 2-4, dialyzing in an acid solution with the pH value of 3-5, filtering for sterilization, and obtaining a filtrate to obtain the purified bovine I type collagen, wherein the cut-off molecular weight of a dialysis bag is 8-100 kDa.

Further, in the step (1), the mass ratio of the non-collagenase proteolytic enzyme to the bovine type I collagen is 0.1-10: 1.

Further, in step (1), the non-collagenase proteolytic enzyme is selected from one or more of pepsin, trypsin and papain. The non-collagenase proteolytic enzyme selectively cleaves the amino-and carboxy-terminal globular regions of the bovine type i collagen molecule, thereby removing the antigenicity of collagen and maintaining the structure and biological activity of the native spatial helical segment of collagen.

Further, in the step (1), the crude treatment method of the bovine type I collagen is an acid extraction method, hydrochloric acid, sulfurous acid, phosphoric acid, sulfuric acid, acetic acid, citric acid or formic acid is added into the bovine bone meal, and the mixture is stirred at 0-25 ℃ to obtain the crude extracted bovine type I collagen.

Further, in step (1), the roughly extracted bovine type I collagen is dissolved with a solution having a pH of 1 to 3.5. The purpose is to provide a suitable pH for the enzyme and to increase the enzyme activity.

Further, in step (1), bovine type I collagen was dissolved at 10 to 37 ℃ at which the enzyme activity was high, the solubility was high and denaturation was not caused.

Further, in step (1), the reaction solution was tested for viscosity using a rheometer to monitor the extent of enzymatic hydrolysis, requiring a small sample size (. about.1 ml) and indicating that the ateptide was substantially complete when the viscosity no longer changed.

Further, in the step (1), enzymolysis is carried out for 12-24 h.

Further, in the step (2), each collagen fraction is separated out and then subjected to centrifugal treatment, and precipitated into telopeptide bovine type I collagen, and the supernatant is collected, subjected to rotary evaporation and concentration, and subjected to alcohol precipitation treatment of the next fraction.

Further, in step (2), the collagen fraction was analyzed for the degree of telopeptide by SDS-PAGE, and it was examined whether the enzyme was substantially removed. Specifically, each collagen fraction was precipitated and a small amount was redissolved in a weak acid and analyzed by SDS-PAGE.

Preferably, in step (2), alcohol solutions with final concentrations of 20-25% v/v, 40-45% v/v, 60-65% v/v and 80-85% v/v are added dropwise in this order.

Further, in the step (2), the alcohol solution is selected from one or more of methanol, ethanol, acetone and isopropanol.

Further, in the step (2), the dropping speed of the alcohol solution is 50-100 ml/h. The collagen, the enzyme and the telopeptide can be better separated by dropwise adding, and the enzyme and the telopeptide are prevented from being wrapped when the collagen is separated out.

Further, in the step (2), an alcohol solution is added dropwise at 1 to 10 ℃. Adjusting the pH of the supernatant to 3.5-5, and reducing the solubility of the bovine type I collagen by adjusting the pH to be high while ensuring the invariance of the collagen; when the temperature is reduced to below 10 ℃, the solubility of the bovine type I collagen is also obviously reduced. The temperature, pH and alcohol are cooperated to separate out the cattle type I collagen without telopeptide, thereby reducing the dosage of alcohol.

Further, after redissolving in an acid solution with the pH value of 2-4, adjusting the pH value to the isoelectric point of the protein, wherein the pH value of the isoelectric point is 5-7, adjusting the isoelectric point and centrifuging to obtain a precipitate, so that a large amount of non-collagenous protease added in the enzymolysis reaction can be further removed, and the high-purity telopeptide-removed bovine I type collagen is obtained; after redissolving, dialyzing in acid solution with pH of 3-5, and adjusting pH to reduce solubility of bovine type I collagen.

Furthermore, the volume ratio of the solution inside and outside the dialysis bag is 0.01-0.5:1, so as to provide concentration difference for dialysis. The molecular weight of the cattle I type collagen purified by the method is 300kDa, the pepsin is about 3kDa, the telopeptide is below 3kDa, a dialysis bag with the molecular weight cut-off of 8-100kDa is adopted, the pepsin, the telopeptide and the acid for adjusting the pH can permeate a semipermeable membrane, and the cattle I type collagen cannot pass through the semipermeable membrane, so that the aim of separating and purifying the cattle I type collagen is fulfilled.

Further, in the step (3), the dialyzed collagen solution is filtered through a 0.45 to 0.22 μm filter, preferably a 0.22 μm filter, and subjected to absolute filtration to remove bacteria.

Further, in step (3), filtration is carried out at 20 to 37 ℃, preferably 30 ℃. The method utilizes the reversible partial helicase behavior of the collagen triple helix under the moderate heating condition, and uses a filter membrane to filter the heated atelocollagen solution, thereby obviously improving the filtering efficiency.

Further, the method also comprises the step of freeze-drying the filtered filtrate to obtain a high-purity telopeptide sterile dried bovine type I collagen product.

The invention claims the cattle type I collagen purified by the purification method.

The invention also claims the application of the bovine type I collagen in preparing food, cosmetics or biomedical materials.

By the scheme, the invention at least has the following advantages:

(1) the invention prepares high-quality sterile collagen from the collagen without the terminal peptide through reasonable and scientific combination of the purification method. Through adjusting pH and temperature, improved I type collagen extraction efficiency of ox and resultant purity simultaneously, reduced the quantity of mellow wine simultaneously, cost reduction.

(2) The invention monitors the process of telopeptide by a viscosity method for the first time. The two ends of the collagen without the decapeptide are provided with non-triple-helical structures, so that the collagen is easy to knot, the viscosity is high, the size of the collagen with only the triple-helical structure after the decapeptide is removed is shorter, the collagen has certain rigidity, and the viscosity is moderately reduced, so that the viscosity change of the collagen solution can effectively reflect the enzymolysis degree, and the collagen is easier to sterilize. When collagen telopeptide is completely hydrolyzed, the viscosity of the reaction solution is not changed any more, and the completeness of telopeptide removal is ensured. Meanwhile, the method is convenient to detect, small in sample usage amount and high in sensitivity.

(3) The purification method breaks through the limitation of the traditional purification method, has wide application range and is suitable for purifying different types of type I collagen.

(4) In the purification process, the removal of the telopeptide achieves the effects of eliminating the immunogenicity of the collagen and improving the biocompatibility, and the purified bovine type I collagen still has protein activity.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is the purified bovine type I collagen of example 1;

FIG. 2 is a graph showing a viscosity change test in example 1;

FIG. 3 is the SDS-PAGE protein electrophoresis chart of the product, raw material and intermediate in example 2;

FIG. 4 shows bovine type I collagen purified by a conventional enzymatic method in a comparative example;

FIG. 5 shows the results of the detection of the isoelectric point in example 1;

FIG. 6 is an infrared spectrum of purified bovine type I collagen from example 1.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

(1) 300mg of bovine type I collagen subjected to hydrochloric acid crude treatment is taken, stirred and dissolved for 8 hours in an acetic acid solution with the pH value of 2.5 according to the concentration of 25mg/ml at 25 ℃, then 60mg of pepsin (the mass ratio of the pepsin to the bovine type I collagen is 0.2:1) is added into the collagen solution, and stirring is continued for 12 hours. Taking 1mL of sample, performing rheological measurement on the solution viscosity (the result is shown in figure 2), indicating that the enzymolysis reaction is finished after the viscosity is stable, then diluting the reaction solution by five times, centrifuging at 9000rpm/min for 15min, and taking the supernatant;

(2) the pH of 60ml of the supernatant was adjusted to 4, 6.7ml of 99% (v/v) aqueous isopropanol (final concentration of 20% (v/v) alcohol) was added dropwise (dropping speed was 60ml/h) at 4 ℃ to precipitate a telopeptide-removed bovine type I collagen fraction 1, which was centrifuged at 10000rpm/min for 15min to obtain a telopeptide-removed bovine type I collagen precipitate fraction 1. Concentrating the supernatant to 50ml, adding 14ml isopropanol aqueous solution (final alcohol concentration is 40% (v/v)) dropwise to precipitate a telopeptide-removed bovine type I collagen fraction 2, centrifuging at 10000rpm/min for 15min to obtain a telopeptide-removed bovine type I collagen precipitate fraction 2. Concentrating the supernatant to 25ml, adding 16ml isopropanol (final alcohol concentration is 60% (v/v)) dropwise to precipitate a telopeptide-removed bovine type I collagen fraction 3, centrifuging at 10000rpm/min for 15min to obtain a telopeptide-removed bovine type I collagen precipitate fraction 3. Concentrating the supernatant to 25ml, dropwise adding 35ml of isopropanol (the final concentration of alcohol is 80% (v/v)), separating out a telopeptide-removed bovine type I collagen fraction 4, performing centrifugation at 10000rpm/min for 15min to obtain a telopeptide-removed bovine type I collagen precipitate fraction 4;

(3) dissolving the atelocillin-removed bovine type I collagen precipitate in acetic acid solution with pH of 4, adjusting to isoelectric point (pH is 6.25, and the result is shown in figure 5), centrifuging at 10000rpm/min for 15min to obtain collagen precipitate, dissolving in acetic acid solution with pH of 3, dialyzing in acetic acid solution with pH of 3 for three days, wherein the cut-off molecular weight of the dialysis bag is 100kDa, and the volume ratio of the solution inside and outside the dialysis bag is 0.1:1, 4 h/time;

(4) adjusting the concentration of the dialyzed collagen solution to 0.06mg/ml, heating to 30 ℃, filtering with a 0.22 mu m filter membrane under the aseptic condition, and freeze-drying the filtrate to obtain 240mg of the atelocollagen-removed aseptic collagen dry product.

Table 1 shows the results of purification obtained by varying pH and/or temperature on the basis of example 1. As can be seen from Table 1, the amount of alcohol used can be reduced and the purity of the product can be improved by adjusting the pH and temperature.

TABLE 1 different purification conditions and product purities

pH of the supernatant	3	4	4
				Temperature of alcohol precipitation	25℃	25℃	4℃
Amount of alcohol used	85ml	80ml	72ml
				Yield of	60％-70％	70-75％	80％-85％

Example 2

(1) Taking 300mg of bovine type I collagen subjected to acetic acid crude treatment, stirring and dissolving the bovine type I collagen in an acetic acid solution with the pH value of 3 according to the concentration of 25mg/ml for 8 hours at 15 ℃, then adding 1500mg of trypsin (the mass ratio of the trypsin to the bovine type I collagen is 5:1) into the collagen solution, and continuously stirring for 20 hours. Measuring the viscosity of the solution by rheology, indicating that the enzymolysis reaction is finished after the viscosity is stable, then diluting the reaction solution by five times, centrifuging at 9000rpm/min for 15min, and taking the supernatant;

(2) the pH of 60ml of the supernatant was adjusted to 4, 6.7ml of 100% (v/v) ethanol aqueous solution (final concentration of alcohol 25% (v/v)) was added dropwise (dropping speed was 100ml/h) at 8 ℃ to precipitate a telopeptide-removed bovine type I collagen fraction 1, and the mixture was centrifuged at 10000rpm/min for 15min to obtain a telopeptide-removed bovine type I collagen precipitate fraction 1. Concentrating the supernatant to 50ml, adding 14ml ethanol water solution (the final concentration of the alcohol is 45% (v/v)) dropwise to precipitate a telopeptide-removed bovine type I collagen fraction 2, centrifuging at 10000rpm/min for 15min to obtain a telopeptide-removed bovine type I collagen precipitate fraction 2. Concentrating the supernatant to 25ml, adding 16ml ethanol (final alcohol concentration is 70% (v/v)) dropwise to precipitate a telopeptide-removed bovine type I collagen fraction 3, centrifuging at 10000rpm/min for 15min to obtain a telopeptide-removed bovine type I collagen precipitate fraction 3. Concentrating the supernatant to 25ml, adding 35ml ethanol (final alcohol concentration is 90% (v/v)) dropwise to precipitate a telopeptide-removed bovine type I collagen fraction 4, centrifuging at 10000rpm/min for 15min to obtain a telopeptide-removed bovine type I collagen precipitate fraction 4. The above collagen fraction 4 was analyzed for the degree of telopeptide by SDS-PAGE, and the telopeptide bovine type I collagen fraction was collected, and the results are shown in FIG. 3.

(3) Dissolving the atelocollagen I precipitate in acetic acid solution with pH of 4, adjusting to isoelectric point (pH is 6.25), centrifuging at 10000rpm/min for 15min to obtain collagen precipitate, dissolving in acetic acid solution with pH of 3, dialyzing in acetic acid solution with pH of 3 for three days, wherein the cut-off molecular weight of the dialysis bag is 50kDa, and the volume ratio of the inner solution to the outer solution of the dialysis bag is 0.5:1, and changing the solution for 4 h/time;

(4) adjusting the concentration of the dialyzed collagen solution to 0.06mg/ml, heating to 30 ℃, filtering with a 0.22 mu m filter membrane under the aseptic condition, and freeze-drying the filtrate to obtain 235mg of the atelocide aseptic collagen dry product.

Comparative example

(1) Soaking appropriate amount of tissue such as pigskin in 0.1mol/L acetic acid for a long time;

(2) separating the tissue residue from the solution containing collagen dissolved from the tissue;

(3) freezing and drying the solution to obtain collagen dry product.

FIG. 1 shows the purified bovine type I collagen of example 1 of the present invention, and FIG. 4 shows the purified bovine type I collagen of comparative example. As can be seen from FIG. 1, the bovine type I collagen of the present invention has an unchanged character, is pure white cotton-like, and still maintains collagen activity. The character of the cattle type I collagen extracted and purified by the traditional enzyme method is slightly yellow, even has granular sensation, and indicates that the purity is not enough.

FIG. 2 shows the results of viscosity measurements of samples at different times during enzymatic digestion in example 1 of the present invention. As can be seen from FIG. 2, the viscosity of the sample at 0h, 6h, 12h and 24h after the enzymolysis, the viscosity of the sample before 12h shows a decreasing trend, and the viscosity does not decrease after 12h, indicating that the enzymolysis is complete.

FIG. 3 is an SDS-PAGE protein electrophoresis chart of the product of example 2 of the present invention during the enzymatic hydrolysis and after purification. As can be seen from FIG. 3, after the sample was subjected to enzymatic hydrolysis, α appeared₂And bands between the enzymes, indicating that excision of the terminal peptide is feasible; the final purified product, the telopeptide and the enzyme, is completely purified, and almost only alpha is stored₁、α₂Band beta, band gamma, indicating that the purified product is mainly left deadThe purity of the peptide collagen is higher.

FIG. 5 shows the results of detection of isoelectric points of collagen in example 1. As can be seen from fig. 5, the collagen isoelectric point in example 1 was pH 6.25.

FIG. 6 is an IR spectrum of collagen of example 1. As shown in FIG. 6, the collagen of example 1 can maintain its characteristic structure after de-telopeptide and purification treatment.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A method for purifying bovine type I collagen is characterized by comprising the following steps:

(2) adjusting the pH value of the supernatant in the step (1) to 3.5-5, sequentially dropwise adding alcohol solutions with final concentrations of 15-25% v/v, 30-45% v/v, 50-70% v/v and 75-95% v/v, sequentially separating out 4 collagen precipitates, and combining to obtain the telopeptide bovine I type collagen precipitate;

2. The purification process according to claim 1, characterized in that: in the step (1), the mass ratio of the non-collagenase proteolytic enzyme to the bovine type I collagen is 0.1-10: 1.

3. The purification process according to claim 1, characterized in that: in step (1), the non-collagenous proteolytic enzyme is selected from one or more of pepsin, trypsin and papain.

4. The purification process according to claim 1, characterized in that: in the step (2), alcohol solutions with final concentrations of 20-25% v/v, 40-45% v/v, 60-65% v/v and 80-85% v/v are sequentially dropped.

5. The purification process according to claim 1, characterized in that: in the step (2), each collagen fraction is separated out and then centrifuged to obtain the telopeptide bovine type I collagen precipitate, and the supernatant is collected, subjected to rotary evaporation and concentration and subjected to alcohol precipitation treatment of the next fraction.

6. The purification process according to claim 1, characterized in that: in step (2), the alcohol solution is selected from one or more of methanol, ethanol, acetone and isopropanol.

7. The purification process according to claim 1, characterized in that: in the step (2), the dropping speed of the alcohol solution is 50-100 ml/h.

8. The purification process according to claim 1, characterized in that: in the step (2), the alcohol solution is added dropwise at 1-10 ℃.

9. A bovine type I collagen purified by the purification method of any one of claims 1 to 8.

10. Use of the bovine type I collagen of claim 9 in the preparation of a food, cosmetic or biomedical material.