TWI435881B - Method of separating collagen from porcine lung and use of product from method - Google Patents

Description

Method for separating collagen from pig lung and application of product

The present invention provides a method for extracting collagen from a mammalian lung.

Pig lungs are mostly positioned as inedible animal by-products. In fact, only when Taiwan's economy is low, some families cut the pig's lungs and cooked them with bamboo shoots. Most of the pig's lungs were slaughtered. Discarded and not used. It is estimated that the weight of the lungs (including the throat and trachea) of a pig at the time of market weight is about 1 kg. If the number of slaughter in Taiwan is about 7.5 million (2005 Taiwan Pig Handbook), it will be nearly 7500 per year. It is a pity that metric tons of pig lungs have not been utilized, and these abandoned pig lungs also bring many environmental problems.

The lungs are composed of several extracellular matrices, including collagen, elastin and proteoglycan. If calculated by dry weight, it contains about 20-30% collagen and 3-5% elastin. These two functions play an important role in supporting the trachea and maintaining vascular stability. Separation of lung collagen is often discussed. Most studies indicate that collagen in the lungs is mostly type 1, third, and fifth. These collagens are widely used in the field of biomedical materials. value. However, since lung tissue contains a high proportion of hemoglobin and cross-linkage with other extracellular matrices, pretreatment and extraction become very difficult and cumbersome, and hemoglobin contamination causes insufficient purity.

Collagen is a high-value biopolymer. Due to its high biocompatibility, it is highly immunogenic and has been paid more and more attention in the biomedical field in recent years. Especially with the characteristics of attracting cell migration, promoting cell proliferation and tissue repair, it is believed that if collagen can be extracted from low-cost pig lungs, it can promote industrial upgrading and improve the economic benefits of the industry.

In view of the above problems, the present invention provides a method for extracting collagen from the lungs of a mammal, which comprises removing mammalian lung impurities and extracting collagen.

The invention collects the lungs of a mammal, and washes the lungs of the mammal through tap water. The washed lungs are placed in crushed ice to cool, remove excess lymph, blood vessels and tracheal tissues and cut the lungs into small pieces, and the treated lungs are The meat grinder is minced, covered with sterile gauze, and the residual moisture is removed. Finally, the treated lung tissue is dispensed and stored frozen.

According to the purpose of the present invention, the method for extracting collagen comprises the steps of: a. adding an extract to a mammalian lung tissue to remove hemoglobin; b. centrifuging and removing the precipitate; c. adding a weak acid solution to the pepsin; d. Centrifuge and collect the supernatant, adjust to pH=7.0 to pH=8.0 with strong alkali for 12 to 72 hours; e. Adjust the supernatant to pH=2.0 to pH=5.0 with hydrogen chloride and add precipitant for salting out 12 Up to 72 hours; f. The supernatant was centrifuged to collect the precipitate and added with weak acid to dissolve and remove the salt; and g. freeze-dried and recovered.

In a preferred embodiment, the mammal is a pig.

In a preferred embodiment, step c of extracting collagen, the precipitate is added with a weak acid solution, and in a more preferred embodiment, the weak acid solution is acetic acid.

In a preferred embodiment, the step d of extracting collagen is adjusted to a pH with a strong base. In a more preferred embodiment, the strong alkaline solution is sodium hydroxide.

In a preferred embodiment, the step f of extracting collagen is followed by the addition of a weak acid. In a more preferred embodiment, the weak acid solution is acetic acid.

In a preferred embodiment, the extract of hemoglobin from the mammalian lung is removed as a liquid for separating membrane proteins. In a more preferred embodiment, the extract comprises tris-hydroxymethylammonium hydrochloride (Tris-HCl). ), polyethylene glycol octyl phenyl ether (Triton X-100) and pH = 8.0 to pH = 10.0.

In a preferred embodiment, its pepsin extraction produces collagen for 12 to 72 hours.

In a preferred embodiment, the temperature of the extracted collagen environment is from 0 ° C to 4 ° C and is centrifuged from 100 g to 11500 g for 30 to 40 minutes.

In a preferred embodiment, the precipitating agent is crystalline sodium chloride.

In a preferred embodiment, the step d of extracting collagen is to de-filter and concentrate. Or dedialysis of the dialysis system.

In a preferred embodiment, the precipitate in step f of extracting collagen and the recovered in step g are collagen, which is suitable for use in biomedical materials or cosmetic technology.

As described above, the main feature of the present invention is a method for extracting collagen from mammalian lungs, which can solve the problem of long-term utilization of mammalian lungs, and can further utilize the collagen prepared by this method for use in biomedical materials. Or cosmetic technology.

The invention may be embodied in different forms and is not limited to the examples mentioned below. The following examples are merely representative of the various aspects and features of the present invention.

Example 1:

Method for extracting collagen from mammalian lungs:

1. Collect the lungs of the mammal and wash the lungs of the mammals with tap water. The washed lungs are placed in crushed ice to cool, remove excess lymph, blood vessels and tracheal tissues and cut the lungs into small pieces. The treated lungs are The meat grinder is minced, covered with sterile gauze, and the residual moisture is removed. Finally, the treated lungs are dispensed and stored frozen.

2. The treated lung is subjected to collagen extraction, a. adding the extract to the mammalian lung tissue to remove hemoglobin; b. centrifuging and removing the precipitate; c. adding the weak acid solution and pepsin to the precipitate; d. centrifuging and collecting The supernatant was adjusted with a strong base to pH=7.0 to pH=8.0 and maintained for 12 to 72 hours; e. The supernatant was adjusted to pH=2.0 to pH=5.0 with hydrogen chloride and a precipitant was added for salting out for 12 to 72 hours. ; f. The supernatant was centrifuged to collect the precipitate and added with a weak acid to dissolve and remove the salt; and g. freeze-dried and recovered.

Example 2:

1. Evaluate the efficiency of four solutions to remove hemoglobin from mammalian lungs. The mechanisms by which these four solutions remove hemoglobin are different.

Solution A is a physiological buffered saline solution.

The B solution was Tris-HCl, pH=8.0 to pH=10.0, and polyethylene glycol octylphenyl ether (Triton X-100).

The C solution was Tris-HCl, pH = 8.0, urea, 2-mercaptoethanol.

The D solution was a 30% ethanol solution.

2. The results are shown in Figure 2. The extraction efficiency of all solvents increased with increasing extraction time. The B solution can be processed for 12 hours to achieve the highest extraction efficiency.

Example 3:

Yield analysis of mammalian lung collagen:

1. The extraction of lung collagen was treated at 4 ° C for 12, 24, 48 and 72 hours respectively. The yield was as shown in Table 1. The yield increased with the extension of the extraction time, and the highest yield was 72 hours. This result shows that it is feasible to isolate collagen from pig lungs.

2. Calculation formula of collagen yield: yield = weight of collagen obtained (g) / original lung tissue weight (kg)

Example 4:

Collagen gel electrophoresis analysis:

1. Collagen SDS-PAGE map As shown in Fig. 3, it can be found from the map that the mammalian lung collagen obtained by the extraction method of the present invention mainly constitutes type I collagen.

2. There is no difference in structure and properties between the collagen obtained in the present invention and the commercial standard, indicating that the extraction technique can indeed separate collagen from the lungs of mammals.

3. The collagen obtained by the present invention has no residual hemoglobin, and it is proved that hemoglobin can be effectively removed by treating with the B solution for 12 hours.

Example 5:

Fourier transform infrared spectroscopy (FTIR):

1. Figure 4 is the effect of different time on the collagen FTIR spectrum of mammalian lungs. The FTIR spectra of the collagen extracted by the four different times obtained in the present invention are the same as those of commercial collagen, and no peak of heat-denatured product (gelatin) is found. , showing its purity and chemical structure is the same as commercial collagen.

Example 6

Thermal denaturation temperature analysis:

Figure 5 shows the effect of different extraction time on the thermal denaturation temperature of mammalian collagen. With the extension of extraction time, the thermal denaturation temperature of collagen also decreases, indicating that its structural integrity is destroyed by pepsin.

Example 7

Capillary electrophoresis analysis:

Capillary electrophoresis separation conditions

(1) BECKMAN COULTER P/ACE ^TM MDQ Capillary Electrophoresis System

(2) Detection wavelength: ultraviolet light 214nm

(3) Sample injection method: air injection, time 6 seconds and pressure difference 2psi

(4) Capillary injection temperature control: 25 ° C

2. The peak of collagen will appear between 8-9 minutes. The detection result is shown in Fig. 6. The absorption spectrum of the collagen subunit is determined by capillary electrophoresis to determine the specific absorption peak, the residual amount of pepsin and whether it is a low molecular weight molecule. produce.

Example 8

Enzyme sensitivity analysis:

1. Figure 7 and Figure 8 are the effects of different extraction times on the sensitivity of mammalian lung collagen enzymes. The higher the value, the more severe the damage by enzymes. Figure 7 is collagenase sensitivity and Figure 8 is trypsin sensitivity. The test shows that the longer the extraction time, the more susceptible collagen is attacked by enzymes and the structural degradation occurs.

A person skilled in the art will readily appreciate that the present invention can be easily accomplished with the results and advantages and those present in the present invention. The method of extracting collagen from mammalian lungs of the present invention is representative of the preferred embodiment, which is exemplary and not limited to the field of the invention. Those skilled in the art will be aware of the modifications and other uses therein. These modifications are intended to be within the spirit of the invention and are defined in the scope of the claims.

The present invention has been described in detail with reference to the embodiments of the present invention, and the invention may be Spirit and scope.

All patents and publications mentioned in the specification are subject to the general skill of the art in the field of the invention. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication is specifically and individually indicated.

The invention as exemplified herein may be practiced in the absence of any element, or a plurality of elements, limitations, or limitations. The nouns and expressions used are as a description and not a limitation of the description, and are not intended to be used to exclude any nouns and expressions that are equivalent to the features or parts thereof shown and described, but Various changes are possible within the scope of the patent application of the invention. Therefore, it is to be understood that the present invention has been disclosed and described herein in accordance with the preferred embodiments and the features of the present invention. Within the scope.

10‧‧‧Lungarian pre-treatment of lungs

11‧‧‧Removing hemoglobin from the lungs

12‧‧‧Enzyme reaction treatment

13‧‧‧ salting out reaction

14‧‧‧ Freeze-drying

Figure 1 is a schematic representation of a method of making collagen in the lungs of a mammal.

Figure 2 is a graph comparing the efficiency of hemoglobin removal from mammalian lungs.

Figure 3 is a gel electrophoresis pattern of collagen.

Figure 4 is a graph of the Fourier transform infrared spectrum of collagen in mammalian lungs at different times.

Figure 5 is a graph showing the thermal denaturation temperature of collagen in mammalian lungs at different times.

Figure 6 is a peak diagram of collagen capillary electrophoresis.

Figure 7 is a graph showing the sensitivity analysis of collagenase.

Figure 8 is a graph of trypsin sensitivity analysis.

10‧‧‧Lungarian pre-treatment of lungs

11‧‧‧Removing hemoglobin from the lungs

12‧‧‧Enzyme reaction treatment

13‧‧‧ salting out reaction

14‧‧‧ Freeze-drying

Claims (7)

A method of extracting collagen from a mammalian lung comprising: removing mammalian lung impurities and performing the following steps: a. adding the extract to the mammalian lung tissue to remove hemoglobin, the extract comprising tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), polyethylene glycol octyl phenyl ether (Triton X-100) and pH=8.0 to pH=10.0; b. Centrifuge and remove the precipitate; c. Add the weak acid solution and pepsin to the precipitate; d. Centrifuge and collect the supernatant, adjust to pH=7.0 to pH=8.0 with strong alkali and maintain 12 to 72 hours; e. The supernatant is adjusted to pH=2.0 to pH=5.0 with hydrogen chloride and a precipitating agent is added for salting out for 12 to 72 hours; f. The supernatant is centrifuged to collect the precipitate and added with weak acid to dissolve and remove the salt. And g. freeze-dry and recover. The method of claim 1, wherein the mammal is a pig. The extraction method according to claim 1, wherein the pepsin reaction extracts collagen for 12 to 72 hours. The method of claim 1, wherein the temperature of the extracted collagen environment is from 0 ° C to 4 ° C, and centrifugation is carried out from 100 g to 11500 g for 30 to 40 minutes. The method of claim 1, wherein the precipitating agent is crystalline sodium chloride. The method of claim 1, wherein the step d is to de-filter the concentration system to remove the salt or the dialysis device. The method of claim 1, wherein the precipitate in the step f and the recovered in the step g are collagen, which is suitable for use in biomedical materials or cosmetic technology.