KR101864816B1 - method for extracting marine collagen from fish skin - Google Patents
method for extracting marine collagen from fish skin Download PDFInfo
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- KR101864816B1 KR101864816B1 KR1020170013921A KR20170013921A KR101864816B1 KR 101864816 B1 KR101864816 B1 KR 101864816B1 KR 1020170013921 A KR1020170013921 A KR 1020170013921A KR 20170013921 A KR20170013921 A KR 20170013921A KR 101864816 B1 KR101864816 B1 KR 101864816B1
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- collagen
- skin
- purification
- pepsin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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- Zoology (AREA)
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- Peptides Or Proteins (AREA)
- Cosmetics (AREA)
Abstract
The present invention relates to a method for extracting marine collagen from a skin, and more particularly, to an extraction method capable of effectively extracting high-purity marine collagen from a fish by-product, which is produced in a fish processing step.
The method of extracting marine collagen from the skin of the present invention includes a pretreatment step of removing scales and foreign substances from a fish by-product, a drying step of drying the pretreated skin in the pretreatment step, a drying step of pulverizing the dried skin in the drying step, A separating step of separating the collagen from the skin powder using an alkali and an acid, and a refining step of refining the collagen separated in the separating step using chromatography.
Description
The present invention relates to a method for extracting marine collagen from a skin, and more particularly, to an extraction method capable of effectively extracting high-purity marine collagen from a fish by-product, which is produced in a fish processing step.
Collagen is the most abundant protein in the animal body, accounting for more than 30% of body protein, and has been found to be at least 19 (Type I-XIX) or more (Nakamura, YN et al. architecture in the M. longissimus thoracis and M. pectoralis profundus from pigs. Meat Science, 64, pp 43-50, 2003). In addition, collagen is the main protein of the connective tissue of an animal, which supports the tissue or organ and surrounds the body, and maintains the body shape. Particularly in vivo, the main constituents of connective tissues such as skin, cartilage and bone are widely distributed in the whole body including 40% in skin, 20% in bones and cartilage, and other blood vessels and organs.
Collagen is a triple helix structure with a diameter of about 14 to 15 nm, a length of 280 to 300 nm, and an average molecular weight of about 300 kDa (Lehninger, AL Biochemistry, 2nd ed., Pp .145, 1975), and (Gly-XY) n, which are covalent cross-linking between molecules in the tropocollagen molecule, the basic unit molecule of fibrous proteins, or tropocollagen molecules (McClain, PE et al., Amino acid composition and cross-linking characteristics of collagen intramuscular connective tissue of striated muscle (Bos taurus). International Journal of Biochemistry, 2 7), pp 121-124, 1971).
Collagen has long been used as a raw material for leather and gelatin, and its applications are becoming more diverse in recent years. In the food industry, edible casing materials are used in meat packaging such as sausage and salami. Collagen used in medicine has been reported to have a therapeutic effect on skin damaged by burns or wounds (Jeyanthi, R. et al., Solid tumor chemotherapy using implantable collagen-poly (HEMA) containing 5-fluorouracil of Pharmacy & Pharmacology, 43, pp.60-62, 1991). As such, collagen is not only used as a functional material for foods and medicines, but also has a function of enhancing the moisture retention of the skin, and is widely used in a variety of fields such as a base material for cosmetics.
Recently, peptides obtained from protein hydrolyzate have been used as potential materials for wrinkle improvement, moisturizing enhancement, elasticity increase and specific skin efficacy through various articles, and a representative example is collagen hydrolyzate.
Collagen hydrolysates, called collagen peptides, are produced by extracting polymer collagen from pork and fish scales, and then hydrolyzing the polymer collagen by a post-treatment such as enzymatic digestion to make it low molecular weight in the form of peptide. In recent years, Collagen products are being sold.
On the other hand, as a byproduct of fishes, food waste has been classified as food waste, and it has been mostly used as compost, like general food. The dried skin has a high utilization rate of 90% or more of proteins and useful substances, so it can be used in a variety of applications such as collagen extraction, cosmetics, health supplements and adhesives. Recently, a variety of products such as collagen-supplemented health supplements and cosmetics have been commercialized, but many depend on imports.
Increasing use of off-site byproducts in the face of competition with Japan, which is one of the world's top seafood consumers, is well suited for economic synergy and eco-friendly industry development by import substitution, high quality fish protein and collagen supply.
In addition, it contains collagen, albumin, myosin, and elastin in a large amount and can be used as high-quality collagen. It can be easily collected in Korea where the consumption of sashimi is high, As the marine fish farming system is steadily being established, it is expected to secure a stable quantity of fish, so it is urgent to evaluate the possibility of effective use of fish byproducts.
It is an object of the present invention to provide an extraction method capable of effectively extracting marine collagen from a fish by-product, which is produced in a fish processing process.
In order to accomplish the above object, a method of extracting marine collagen from a skin of the present invention comprises: a pretreatment step of removing scales and foreign substances from a fish by-product; Drying the pre-treated skin in the pre-treatment step; A crushing step of crushing the dried article in the drying step to obtain an artificial powder; A separation step of separating collagen from the skin powder using an alkali and an acid; And separating the collagen separated in the separating step by chromatography, wherein the separating step comprises the steps of: a) obtaining an alkaline residue from which non-collagen protein has been removed by adding a sodium hydroxide solution to the skin powder; B) adding an acetic acid solution to the alkaline residue and stirring the mixture, separating the supernatant with a centrifugal separator to extract acid-solubilized collagen, and c) adding pepsin to the acid-solubilized collagen and stirring, separating the supernatant with a centrifuge Followed by the addition of a sodium chloride solution to precipitate the precipitate, which is then dialyzed with distilled water to extract pepsin-solubilized collagen.
In the pretreatment step, the skin is washed with water to remove foreign matter, and then the solution is added to a 0.1 to 0.2M sodium hydroxide solution and agitated for 6 to 24 hours to remove the scales attached to the skin.
The purification step is carried out using ion exchange chromatography equipped with a column packed with cellulose phosphate.
In the purification step, the pepsin solubilized collagen was dialyzed against 20 mM Na 2 HPO 4 to inactivate pepsin, dialyzed against 50 mM acetic acid containing 2 M components, and the fraction eluted by the ion exchange chromatography was dialyzed against 0.5 M acetic acid Solution, dialyzed with distilled water, and lyophilized.
The purification step is purified stepwise using multiple chromatographies.
In the purification step, the pepsin solubilized collagen is desalted by gel filtration chromatography, followed by primary purification by ion exchange chromatography, secondary purification by hydrophobic interaction chromatography, and tertiary purification by gel filtration chromatography.
In the purification step, the pepsin solubilized collagen is first purified by hydrophobic interaction chromatography, followed by secondary purification by ion exchange chromatography, and then by tertiary purification by gel filtration chromatography.
As described above, according to the present invention, the skin separated from a fish is dried at a low temperature to remove water, and then processed into a powder state to extract marine collagen, thereby preventing deterioration of the skin and preventing thermal denaturation of collagen, .
In addition, the present invention can further effectively purify marine collagen with high purity by performing purification using chromatography. The purified marine collagen retains the collagen structure of the polymer, which is not a low molecular weight collagen peptide, and has a gel-forming ability and an excellent moisturizing effect, so that it can be usefully used for various purposes.
Figs. 1 and 2 show the results of measuring the ratio of acid-solubilized collagen (ASC)
FIG. 3 is a result of measuring the heat denaturation temperature of acid-solubilized collagen (ASC) isolated from the flounder,
FIGS. 4 to 7 are the results of SDS-PAGE analysis of RS-AL and ASC extracted from the skin and the skin of each species,
FIG. 8 shows the results of SDS-PAGE analysis of high purity marine collagen isolated from pepsin-solubilized collagen.
Hereinafter, a method for extracting marine collagen from a skin according to a preferred embodiment of the present invention will be described in detail.
The method of extracting marine collagen from the skin according to an embodiment of the present invention includes a pretreatment step of removing scales and foreign substances from the fish by-product, a drying step of drying the pretreated skin in the pretreatment step, A step of grinding to obtain an artificial powder; a step of separating the collagen from the artificial powder using alkali and acid; and a purification step of purifying collagen separated in the separation step using chromatography. Hereinafter, the steps will be described.
1. Pre-processing step
The pretreatment step removes scales and foreign matter from the fish byproduct.
The skin is the skin of a fish, and any skin of the fish can be applied, but it is preferably one selected from olive flounder, oyster, perch and sea buckthorn. Four kinds of fish are consuming a lot, and it is easy to supply the relative. In addition, salmon shells can be used as skin.
Since the skin is mainly produced in the fish processing process, it is washed 2 or 3 times with clean water to remove various foreign matter and blood in the skin.
After washing with water, the next drying step can be carried out, but the scales attached to the skin are preferably removed before drying. When the grinding process described below is performed without removing the scales, the pulverizer discharge port is clogged due to the scales, so that it takes a lot of time in the pulverizing operation and the parts of the pulverizer discharge part are broken due to the increase of the pulverizing water pressure There are various problems such as the occurrence of the phenomenon. Therefore, it is necessary to remove scales attached to the skin. If the scales are removed manually by the operator using the tool, the process will be difficult and time consuming.
Therefore, the present invention uses a sodium hydroxide solution so as to simply and effectively remove scales attached to the skin. For example, the skin, which has been washed with water and the foreign substance is removed, is put into a 0.1 to 0.2M sodium hydroxide solution and agitated for 6 to 24 hours to remove the scales attached to the skin. Agitation in the sodium hydroxide solution will effectively remove scales, although there is some difference depending on the fish species. In the 0.1 M sodium hydroxide solution, 80 to 90% of the scale is removed, and in the 0.2 M sodium hydroxide solution, 90 to 100% of the scale is removed. Remove the scale and rinse again with water.
In addition, the speed of scales removal can be shortened by applying an ultrasonic wave to the sodium hydroxide solution contained in the stirring tank and stirring it. For example, an ultrasonic vibrator may be installed on the bottom of the stirring tank and stirred while applying ultrasonic waves of 40 to 60 kHz into the stirring tank. Ultrasonic vibration is applied to the skin in a state in which the adhesion force of the scales is weakened by the sodium hydroxide solution so that the scales can be more easily removed.
As described above, according to the present invention, scales can be very easily removed by using a sodium hydroxide solution, so that clogging of the crusher discharging part is not caused at the crushing of the crusher, and the breakage of the crusher parts due to clogging does not occur. And the manual operation for removing the scales can be omitted, thereby shortening the manufacturing time and reducing the cost and manpower.
2. Drying stage
Next, the pre-treated skin is dried in the pretreatment step. The extraction yield of collagen is increased and dried for prevention of deterioration and crushing. In the drying step, the skin is dried to a moisture content of 2-7% by weight. A freeze-drying method and a hot-air drying method can be applied. Preferably, a freeze drying (FD) method is used to prevent denaturation of the collagen.
Rapidly frozen at a temperature of -50 to -40 DEG C for 10 to 20 hours, and then dried at about -40 DEG C for 48 hours in a freeze dryer having a degree of vacuum of 0.1 to 0.5 torr. It is needless to say that the conventional freeze-drying method applicable to the manufacture of foods can be applied.
If the lid is freeze-dried, since the moisture is removed by sublimation in the frozen state, the dried product has a light porous structure and maintains its original shape and size, and is treated at a low temperature without applying heat, Migration of soluble components in drying, nonenzymatic browning, protein denaturation, etc. hardly occurs.
In the case of hot air drying, the skin can be dried by hot air at 30 to 80 ° C. The temperature of the hot air is 65 DEG C or less, preferably 40 to 60 DEG C in order to prevent the collagen from being denatured at the time of hot air drying. The collagen contained in the skin has a higher denaturation temperature than the separated state.
3. Crushing step
The dried skin is pulverized to an appropriate size using a pulverizer to obtain an artificial powder. For example, 50 to 150 mesh size.
4. Separation step
The collagen is separated from the skin powder after obtaining the skin powder.
The step of separating may include, for example, a) a step of adding a sodium hydroxide solution to the skin powder to obtain an alkaline residue from which non-collagenous protein has been removed, b) a step of adding acetic acid solution to the alkaline residue and stirring the supernatant, C) adding pepsin to the acid-solubilized collagen, stirring the mixture, separating the supernatant with a centrifuge, adding a sodium chloride solution, precipitating the precipitate, dialyzing with distilled water to extract pepsin-solubilized collagen .
The step of obtaining an alkaline residue from which a non-collagenous protein has been removed by adding sodium hydroxide solution to the powder of the skin is specifically as follows.
The bulk powder and the sodium hydroxide solution were mixed at a weight ratio of 1: 5 to 10, and then the mixture was stirred at room temperature (20 to 25 ° C) for 12 to 24 hours. Then, an alkaline residue was obtained by removing the non-collagenous protein using a centrifuge can do.
The step of extracting acid-solubilized collagen by adding acetic acid solution to the alkaline residue is as follows.
After washing the alkaline residue with distilled water, add acetic acid solution to extract the collagen. The alkali residue and the acetic acid solution are mixed at a weight ratio of 1: 5 to 10, and the mixture is stirred at room temperature (20 to 25 ° C) for 12 to 24 hours, and then the supernatant is separated using a centrifuge to obtain acid-solubilized collagen.
The step of extracting pepsin-solubilized collagen by adding pepsin to the acid-solubilized collagen is as follows.
Pepsin is added to the acid-solubilized collagen and stirred for 10 to 20 hours. The supernatant is separated with a centrifuge, and 2M sodium chloride solution is added to precipitate the precipitate. The precipitate is dialyzed with distilled water to obtain pepsin-solubilized collagen.
5. Purification step
The pepsin solubilized collagen separated in the separation step can be purified by chromatography to obtain high purity marine collagen.
As an example, the purification step can be purified using ion exchange chromatography equipped with a column packed with cellulose phosphate.
Pepsin-solubilized collagen was dialyzed against 20 mM Na 2 HPO 4 to inactivate pepsin, dialyzed against a 50 mM acetic acid solution (pH 4.8) containing 2 M urea, and then eluted with ion-exchange chromatography . Purification was carried out with a linear gradient (60 ml / h) of 0 to 600 mM NaCl in a column filled with cellulose phosphate (P11, Whatman, Maidstone, UK), and the fraction eluted at 230 nm was diluted with 0.5 M acetic acid Solution, dialyzed with distilled water, and lyophilized to obtain high purity marine collagen.
On the other hand, as another example of the purification process, purification can be performed stepwise using a plurality of chromatographies. Examples of usable chromatography include ion-exchange chromatography, gel filtration chromatography, and hydrophobic interaction chromatography.
For example, peptidyl-solubilized collagen is desalted by gel filtration chromatography, followed by primary purification by ion exchange chromatography, second purification by hydrophobic interaction chromatography, and third purification by gel filtration chromatography. I can refine it.
In another example, the pepsin solubilized collagen is first purified by hydrophobic interaction chromatography, followed by second purification using ion exchange chromatography, and then third purification using gel filtration chromatography.
Hereinafter, the contents of the present invention will be described in detail with reference to the following experimental examples. It is to be understood that the scope of the present invention is not limited to the following examples.
<Examples>
After the scales were removed, the fishes were quickly frozen at -45 ° C for 15 hours and then frozen at -40 ° C for 48 hours in a freeze dryer with a degree of vacuum of 0.5 torr. Dried and pulverized to prepare an artificial powder. Then, 0.1 M sodium hydroxide solution was added at a weight ratio of 10 times and the mixture was stirred at room temperature (20 ° C.) for 16 hours. Then, an alkaline residue (RS-AL) from which non-collagen protein was removed was centrifuged .
The resulting alkaline residue was washed with distilled water and mixed with a 0.5 M acetic acid solution at a weight ratio of 10 times. The mixture was stirred at room temperature (20 ° C) for 16 hours, and the supernatant was separated using a centrifuge to obtain acid-soluble collagen ). (EC 3.4.23.1; crystallized and lyophilized, Sigma, MO) was added to the acid-solubilized collagen, and the mixture was stirred for 14 hours. The supernatant was separated by a centrifuge, and 2M sodium chloride solution was added thereto. The precipitate was dialyzed with distilled water Pepsin solubilized collagen was obtained.
Pepsin solubilized collagen was dialyzed against 20 mM Na 2 HPO 4 to inactivate pepsin and dialyzed against 50 mM acetic acid solution (pH 4.8) containing 2 M urea. Cellulose phosphate (P11, Whatman, Maidstone, UK) , The fraction eluted at 230 nm was dialyzed against a 0.5 M acetic acid solution containing 2.0 M NaCl and recovered. The extract was dialyzed against distilled water, and freeze-dried To obtain high-purity marine collagen.
1. Proportion of acid soluble collagen (ASC) in the skin
In order to investigate the proportion of ascites acquired in each species, the dialysed ASC was lyophilized in 1 ml of 1.5 ml tube and weighed. Weighed before and after freeze drying in 1.5 ml tube, The percentages of ASCs extracted in the api were calculated by the following equation.
S = {(Y1 x Y2) / X} x100
S:% ASC in the affair
Y1: Weight of dialysate precursor (g)
Y2: ASC ratio (%) for dialysis lyophilized gel
X: Wet weight of the first used skin (g)
The experimental results are shown in Fig.
1, the percentage of acid-soluble collagen (ASC) in flounder, oyster, perch, and red sea bream was 9.8%, 8.01 ± 0.19% and 5.75%, respectively, Showed the lowest value of 2.69%.
Compared to the collagen content of four tibia fish species, there was a large difference between species and showed a tendency to support the histological observation, but the value of rainbow trout was relatively low.
ASC collagen ratios were analyzed using a Sircol TM Soluble Collagen Assay kit (Biocolor, UK). The calibration curve of the standard solution was prepared and the collagen content of the sample to be analyzed was determined and shown in FIG.
The collagen content of the skin was measured by using the collagen assay kit. The content of collagen was 9.73% for flounder, 3.46% for fish, 6.96% for perch and 11.83% for sea bream.
2. Apical collagen thermodynamic stability study
The heat denaturation temperature was measured at a constant heating rate (0.5 ° C / 1 min) for acid-solubilized collagen isolated from flounder skin using Micro DSC (Setaram, France). At this time, ASC of tail vertebrae The heat denaturation temperature was measured by the same method and compared with the data of fish, and it is shown in FIG.
The heat denaturation temperature was measured at a constant heating rate (0.5 ° C / 1 min) using a Micro DSC (Setaram, France). At the same time, a tail vertebra ASC (Sigma Aldrich, USA) (Fig. 3: ∘, ) was 12 ° C lower than that of the rat vertebrate collagen (Fig. 3: ▴).
Suggesting that low denaturation temperature is due to low proline hydroxylation of fish collagen. In addition, the flounder skin ASC showed a higher resistance to thermal deformation than the rainbow trout muscle ASC due to the higher temperature of 3.9 ℃.
3. Molecular characterization
The molecular characterization of the collagen was analyzed by the amino acid composition and SDS-PAGE analysis.
0.5 ml of acid-solubilized collagen was weighed into an 18 ml test tube, 3 ml of 6N HCl was added, and the test tube was sealed with a vacuum pump. The sealed test tube was hydrolyzed in a heating block at 121 ° C for 24 hours, then acid was removed with a rotary evaporator at 50 ° C and 40 psi, followed by 10 ml with a sodium loading buffer. Ml was taken and filtered through a membrane filter (0.2 ㎕) and quantitatively analyzed with an amino acid analyzer (S-433H, SYKAM GmbH, Germany). The column size was 4.6 × 150 mm, the column temperature was 57-74 ° C., the buffer flow rate was 0.45 mL / min and the flow rate of the reagent was 0.25 (LCA K06 / Na) ㎖ / min. The buffer pH range was 3.45 ~ 10.85 and the wavelength was 440nm and 570nm.
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed according to the method of Laemmli (1970).
Each sample was adjusted to a concentration of 1 mg / ml in Sample buffer (50 mM Tris-HCl, pH 7.5; 50% glycerin, 1% SDS, 0.02% bromophenol blue, BPB) and heat denatured by heating at 95 ° C for 5 minutes The sample was allowed to stand at room temperature for 10 minutes to prepare a sample. The prepared samples were prepared with 7.5% gel using 40% polyacrylamide consisting of 3% stacking gel and 7.5% separate gel. The electrophoresis was performed using Bio-RAD Power Pac Basic (USA) 35 mA / gel. The protein bands were stained in four steps according to Fairbanks et al., (1971), in which the staining solution of Coomassie brilliant blue (CBB), 2-propanol and acetic acid was staged in a total of 4 steps. Were performed for 2 hours each. At this time, the marker used to confirm the molecular weight of the sample was SDS-PAGE Molecular Weight Stadards (Bio-Rad Laboratories, High range, USA).
SDS-PAGE results of each species are shown in FIGS. 4 to 7, respectively. FIG. 4 is an SDS-PAGE pattern of RS-AL and ASC extracted from a flounder and a flounder, FIG. 5 is an SDS-PAGE pattern of RS-AL and ASC extracted from a right and left skeleton, FIG. FIG. 7 is an SDS-PAGE pattern of RS-AL and ASC extracted from the Japanese black bulb skin and the Japanese black bulb skin. 4 to 7, MP means a maker protein.
The SDS-PAGE of each species was composed of subunit α1 (I), α2 (I) and its equivalent β-chain, and two polymer bands were found on the β-chain. This shows SDS-PAGE patterns of typical type I collagen, and molecular weights confirm that α1 (I) and α2 (I) protein bands are formed slightly higher than 116.2 k in both AFI and RS-AL and ASC And β-chain protein bands were mainly distributed around 200k.
The results of analysis of constituent amino acids are shown in Table 1 below.
(mg / 100g)
Table 1 shows the resultant amino acid content of acid-soluble collagen (ASC) extracted from the flounder, oyster, sea bass, and red sea bream.
The total amino acids of the fish were 70.15g / 100g, 70.67g / 100g, 74.02g / 100g, and 67.41g / 100g, respectively, and the amino acids of the sea bass were the highest in the olive flounder, Glycine, which can characterize the repetitive Gly-XY amino acid sequence, accounted for about 25% of 17.64g / 100g, 17.52g / 100g, 18.37g / 100g and 17.20g / 100g, respectively. Proline was 10.86g / 100g , 10.53 g / 100 g, 12.46 g / 100 g, and 10.50 g / 100 g, respectively.
4. Analysis of high purity marine collagen
The results of SDS-PAGE analysis of high-purity marine collagen isolated from pepsin-solubilized collagen are shown in FIG. Collagen was detected in high purity marine collagen of four species fish. Collagen was composed of α1 (I) and α2 (I) subunits such as ASC and β-chain of the same, and two polymer bands were identified on the β-chain. 11: MP: marker protein; A: Flounder; B: Right bank; C: perch; D: It means red snapper.
Collagen contains hydroxyproline and hydroxylysine, which are hydroxylated amino acids, and a repeating amino acid sequence of Gly-XY at the triple helical region. Proline and hydroxyproline are located at the positions of X and Y, The presence of collagen is confirmed by glycine, proline and hydroxyproline contents.
Table 2 shows the results of analyzing amino acid residues in order to confirm the content of glycine, proline, hydroxyproline and the like for high purity marine collagen.
Constituent amino acid
(mg / 100g)
As a result of analyzing amino acid residues, glycine 353 (35%), hydroxyproline 75 (7%) and proline 84 (95%) were dissolved in 1,000 residue to confirm the content of glycine, proline, hydroxyproline and the like on high purity marine collagen. 8%), hydroxylysine 9 (1%) and so on.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation. Accordingly, the true scope of protection of the present invention should be determined only by the appended claims.
Claims (7)
Drying the pre-treated skin in the pre-treatment step;
A crushing step of crushing the dried article in the drying step to obtain an artificial powder;
A separation step of separating collagen from the skin powder using an alkali and an acid;
And a purification step of purifying the collagen separated in the separation step by using chromatography,
Wherein the separation step comprises the steps of: a) adding a sodium hydroxide solution to the skin powder to obtain an alkaline residue from which a non-collagen protein has been removed; b) adding an acetic acid solution to the alkaline residue and stirring, separating the supernatant with a centrifuge C) extracting the supernatant with a centrifuge after adding pepsin to the acid-solubilized collagen, c) separating the supernatant with a centrifuge, and then precipitating the precipitate with sodium chloride solution by dialysis with distilled water to extract the pepsin-solubilized collagen Including,
In the pretreatment step, the skin is washed with water to remove foreign matters, and then the solution is added to a 0.1 to 0.2M sodium hydroxide solution contained in a stirring tank, stirred for 6 to 24 hours to remove scales attached to the skin,
Wherein the pre-treatment step is carried out while stirring ultrasonic waves of 40 to 60 kHz into the stirring tank.
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| CN112891609A (en) * | 2021-01-15 | 2021-06-04 | 蚌埠学院 | Porous material taking sheepskin as raw material and preparation method and application thereof |
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| CN113943770B (en) * | 2021-11-18 | 2023-04-07 | 中国海洋大学 | Fish skin collagen and extraction method thereof |
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