CN103320485A - Preparation method of fish-skin collagen for medical biomaterial - Google Patents
Preparation method of fish-skin collagen for medical biomaterial Download PDFInfo
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Abstract
The invention provides a preparation method of fish-skin collagen for a medical biomaterial. The preparation method provided by the invention comprises steps of raw material treatment, collagen extraction, extract purification and the like. By the adoption of the preparation method of fish-skin collagen for a medical biomaterial, non-collagen components of fish-skin can be fully and effectively removed; high-purity collagen with the content of obtained hydroxyproline being greater than or equals to 9%, the content of non-collagen being less than or equals to 1% and the content of ash being less than or equals to 1% is obtained; and fish resources can be utilized within a larger range. The preparation method has advantages of simple technology, easy operation, short processing time and obvious biocompatibility effect, and is suitable for industrial production.
Description
Technical Field
The invention belongs to the field of preparation of biomedical materials, and particularly relates to a method for extracting natural polymer collagen serving as a biomedical material from fish skin.
Background
Collagen is the most abundant important structural protein in animals, widely distributed in skin, bone, cartilage, teeth, tendons, ligaments and blood vessels of all multicellular animals from aquatic animals to vertebrates, is the main component of supporting tissues and connective tissues, accounts for about 25 percent of the total protein of organisms, and has multiple functions of supporting organs, protecting the organisms, connecting, nourishing and the like in the animals.
The majority of mammalian collagen fibers belong to type I collagen consisting of 2 strands of alpha1Peptide chain and 1. alpha2The peptide chain forms a triple helix supercoiled structure through an alpha helix structure. This unique structure gives it many useful features: high tensile strength, biodegradability, promotion of cell growth, and the like. Is one of important natural polymer materials in the aspects of clinical application as hemostatic materials, soft tissue filling materials and the like. In more than 10 years, with the rapid development of molecular biology, genetics, material application and other disciplines, the properties and biological functions of collagen are more deeply known and understood, the application range of collagen is greatly expanded, and the application of collagen in the fields of biomedical materials, tissue engineering related biomaterials and the like is particularly concerned.
The natural high molecular collagen has wide source, and can be obtained from skin, bone, tendon, etc. of terrestrial animals such as pig and cattle. It can also be obtained from aquatic animals such as fish skin/scale. Because epidemic diseases of the land animals frequently occur, mad cow disease, foot and mouth disease and other diseases, the land animals are threatened to live, and the products of the land animals also cause the risk of zoonosis to human beings. Aquatic animals are receiving much attention because of their safety and lower antigenicity. The fish collagen is mainly enriched in fish skin and fish scales, particularly the collagen in the fish skin accounts for about 70 percent of the total protein of the fish skin, belongs to type I collagen, and is a high-quality material for preparing biomedical materials.
The fish skin is often treated as a waste resource in the process of processing aquatic products, and cannot be well and fully utilized. According to statistics, the amount of abandoned fish skin in the processing process reaches tens of thousands of tons every year, which not only pollutes the environment, but also wastes resources.
In the prior art, the fish skin of deep-sea fish is mainly utilized, even the fish skin of specific fish can be processed to obtain the fish skin collagen for the medical grade biological material, the operation steps are complicated, the processing time is long, the content of the obtained fish skin collagen is low, the ash content is high, the biocompatibility is not good, and the application range is narrow. In the research literature on extracting collagen by using fish skin disclosed at home and abroad at present, a processing technology for pretreating raw materials by using EDTA also exists, the pollution of the production process is serious, the extraction period is long, only the pretreatment technology needs at least one week, and the industrialization is difficult to realize.
The fish species are very rich, and the fish lives in different environments of seawater and fresh water, so that the structure of the fish skin is different, and no report about a complete and effective method for industrially preparing the fish skin collagen for the medical grade biomaterial is found so far.
Disclosure of Invention
Aiming at the problems in the preparation of fish skin collagen for medical grade biomaterial, the invention provides a novel preparation method.
The invention provides a preparation method of fish skin collagen for medical grade biomaterial, which comprises the following steps:
1) raw material treatment: mincing the skin of the cleaned and dehydrated marine fish or freshwater fish into 0.5-5 cm2Adding 2-5% NaCl salt solution with the material-liquid ratio of 1:30 (w/w), stirring at 0-4 ℃ for 8-12 hours, discharging waste liquid, repeating for 3-4 times, cleaning with deionized water, and putting the cleaned fish skin into 0.05-0.2M NaOH solution with the material-liquid ratio of 1:30 (w/w)Stirring for 32-48 hours, replacing the NaOH solution once every 8-12 hours, and fully cleaning the fish skin treated by the alkali solution by using deionized water until the fish skin is neutral;
2) extracting collagen: putting the cleaned fish skin into 0.2-1M acid solution according to the material-liquid ratio of 1: 10-30 (w/w), mashing, stirring for 4-8 hours, centrifuging, taking supernatant to obtain acid-soluble collagen solution, repeating for 2-3 times, combining the centrifuged supernatant, adding 0.1-1% of protease, stirring for 24-60 hours, and performing enzymatic treatment to obtain enzymatic hydrolysis collagen solution;
3) and (3) purifying an extract: adding saturated NaCl solution until the salt content in the collagen liquid reaches 2-5%, standing for 2-6 hours, centrifuging to obtain precipitated crude collagen, dissolving the crude collagen in pure water according to the material-liquid ratio of 1: 5-10 (w/w), dialyzing for 4-6 times by using a semipermeable membrane, purifying the collagen liquid, and carrying out freeze drying treatment at-60 ℃ to obtain the natural high-molecular fish skin collagen for the medical-grade biomaterial, wherein the water content of the natural high-molecular fish skin collagen is less than or equal to 14%.
Preferably, the fish skin adopted in the step 1) is fresh or frozen various sea fish or freshwater fish skin;
preferably, the sea fish skin adopted by the fish skin collagen preparation method is cod skin, sole skin, squid skin and salmon skin, and the fresh water fish skin is tilapia skin, black carp skin, grass carp skin, silver carp skin and bighead carp skin;
preferably, the step of washing the fish skin in the step 1) is to remove impurities such as fish scales, meat residues and fat from fresh or frozen sea fish or freshwater fish, wherein the ratio of the fish skin to the feed liquid is 1: stirring with 20-30 (w/w) 0-4 ℃ pure water, cleaning for 30 minutes, removing wastewater, and repeating once;
preferably, the step of treating the fish skin to neutrality in step 1) is: rinsing with deionized water for 4 times, wherein each time lasts for 0.5-1 hour, and the pH is 7-8;
preferably, the temperature of the deionized water in the step 1) is 0-4 ℃, and the feed-liquid ratio of the fish skin to the deionized water is 1:30 (w/w);
preferably, the acidic solution in step 2) is an acetic acid, phosphoric acid or citric acid solution;
preferably, the protease in step 2) is pepsin, trypsin or alkaline protease;
preferably, the step of dialysis of the semipermeable membrane in the step 3) is: the molecular weight cut-off of the semipermeable membrane is 100KD, the dialyzed external solution is pure water with the material-liquid ratio of 0-4 ℃ being 1: 20-40 (w/w), the dialyzate is replaced once every 8-12 hours, and the process is repeated for 4-6 times;
preferably, the preparation steps of the fish skin collagen are all carried out at the temperature of 0-4 ℃.
The invention has the beneficial effects that:
1) the non-collagen components of the fish skin can be sufficiently and effectively removed, and the high-purity collagen with the hydroxyproline content of more than or equal to 9 percent, the non-collagen content of less than or equal to 1 percent and the ash content of less than or equal to 1 percent is obtained;
2) the method is suitable for preparing collagen of the fish skin of marine fishes, also suitable for preparing collagen of the fish skin of freshwater fishes, can adopt fresh fish skin and frozen fish skin, can solve the problem of diversity of fish varieties and can utilize fish resources in a wider range;
3) the process is simple and easy to operate, has short processing time and is suitable for industrial production;
4) good biocompatibility, and is superior to the basic requirement of low antigenicity of biomedical materials.
Drawings
The various aspects of the present invention will become more apparent to the reader after reading the detailed description of the invention with reference to the attached drawings. Wherein,
FIG. 1 shows a process flow diagram for the preparation of fish skin collagen for medical grade biomaterials according to the present invention;
FIG. 2 shows an SDS-PAGE pattern of fish skin collagen according to the invention. The numbers below the symbol M are the protein molecular weight markers in Kilodaltons (KD). Symbol a is Bovine Serum Albumin (BSA), symbol B is Marker (Marker), and symbol C is fish skin collagen. Band 1 is a hetero-protein, band 2 is a trimer, band 3 is a dimer, band 4 is α 1, and band 5 is α 2.
Detailed Description
In order to more clearly understand the technical contents of the present invention, embodiments of the present invention are described in further detail below with reference to the accompanying drawings.
Fig. 1 shows a process flow diagram for preparing fish skin collagen for medical grade biomaterial according to the present invention, and referring to fig. 1, the present invention uses fresh or frozen sea fish or freshwater fish skin, the sea fish skin can be cod skin, sole skin, squid skin, salmon skin, etc., and the freshwater fish skin can be tilapia skin, black carp skin, grass carp skin, silver carp skin, bighead carp skin, etc. After removing sundries such as fish scales, residual meat, fat and the like attached to the fish skin, mixing the fish skin with the fish skin in a feed-liquid ratio of 1: stirring with 20-30 (w/w) 0-4 ℃ pure water, cleaning for 30 minutes, then discharging wastewater, cleaning for 2 times, and dehydrating. (S1)
Mincing fish skin into 0.5-5 cm2Adding a 2-5% NaCl salt solution with a material-liquid ratio of 1:30 (w/w), stirring for 8-12 hours at 0-4 ℃, then discharging waste liquid, repeating for 3-4 times, rinsing for 4 times with 0-4 ℃ deionized water (the material-liquid ratio is 1:30 w/w), 0.5-1 hour each time, (S2) stirring the fish skin with 0-4 ℃ 0.05-0.2M NaOH (the material-liquid ratio is 1:30 w/w) for 32-48 hours, replacing the solution once every 8-12 hours, rinsing for 4 times with 0-4 ℃ deionized water (the material-liquid ratio is 1:30 w/w), 0.5-1 hour each time, and adjusting the pH to 7-8. (S3) the fish skin is put into 0 to 4 ℃ 0.2 to 1M acetic acid, phosphoric acid or citric acid solution (the material-liquid ratio is 1:10 to 30 w/w) to be mashed, stirred for 4 to 8 hours and centrifuged at 0 to 4 ℃ and 10000 to 15000rpmTaking supernatant, placing the precipitated fish skin in 0-4 ℃, 0.2-1M acetic acid, phosphoric acid or citric acid solution (the material-liquid ratio is 1: 20-30 w/w) for stirring, repeating for 2-3 times, (S4) combining the supernatant, adding 0.1-1% of pepsin, trypsin or alkaline protease, carrying out enzymolysis for 24-60 hours at 0-4 ℃ (S5), adding saturated NaCl solution, stirring until the salt concentration of the solution is 2-5%, standing for 2-6 hours, centrifuging at 0-4 ℃ and at 10000-15000 rpm, collecting the precipitated collagen, (S6) dissolving the collagen in pure water (1: 5-10 w/w), dialyzing for 48-72 hours, the molecular weight cut-off of the semipermeable membrane is 100KD, the dialyzing external liquid is pure water with a material-liquid ratio of 1: 20-40 (w/w) at 0-4 ℃, the dialyzing liquid is replaced once every 8-12 hours, and the process is repeated for 4-6 times. And finally, freeze-drying the purified collagen solution in a low-temperature freeze dryer at the temperature of 60 ℃ below zero until the water content is below 14 percent to obtain the natural high-molecular fish skin collagen for the medical-grade biological material. (S7)
Example 1
200g of fresh cod skin is taken to remove the residual meat and fat, cut into small pieces of 1cm multiplied by 1cm by a slicer, rinsed with 20 times of pure water at 4 ℃ for 30 minutes, repeated once and drained. The fish skin is put into 3 percent NaCl solution at 4 ℃ with the material-liquid ratio of 1:30 (w/w), stirred for 40 hours, changed every 10 hours, and drained. The mixture was washed with deionized water at 4 ℃ for 30 minutes 4 times at 1:20 (w/w) and the water was drained.
The fish skin is put into 0.2M NaOH solution at 4 ℃, the ratio of the materials to the liquid is 1:30 (w/w), the stirring is carried out for 40 hours, the liquid is changed every 10 hours, and the draining is carried out. Washing with deionized water at 4 deg.C (1: 30 w/w) for 3 times (1 hr each time) to pH 7-8, and draining off water.
Putting the cleaned fish skin into 0.5M phosphoric acid solution at 4 ℃, stirring the mixture at a material-liquid ratio of 1:30, centrifuging the mixture at 4 ℃ by 10000rpm, and taking supernatant for later use. The centrifuged precipitate was further added with 0.5M phosphoric acid solution, and triturated with stirring at a feed-to-liquid ratio of 1:20 (w/w) for 3 hours. Centrifuging at 4 deg.C at 10000rpm, and collecting supernatant. Adding 0.5M acetic acid solution into the centrifuged precipitate, stirring at a ratio of 1:20 (w/w) for 4 hr, centrifuging at 4 deg.C at 10000rpm, collecting supernatant, mixing the supernatants for 3 times, adding 0.2% pepsin, and continuously stirring at 4 deg.C for 60 hr.
Adding saturated NaCl solution to salt out to make NaCl concentration in the feed liquid be 3%, precipitating and standing. And centrifuging the salting-out solution at a low temperature of 4 ℃ at the rotating speed of 10000rpm, and collecting the precipitated crude collagen. Dissolving crude collagen at a ratio of 1:10 (w/w) in pure water at 4 deg.C, dialyzing with pure water at a ratio of 1:20 (w/w) and semipermeable membrane with molecular weight cutoff of 100KD at 4 deg.C for 48 hr, and replacing the dialyzed solution every 8 hr.
Collecting the dialyzed external solution, and freeze-drying in a low-temperature freeze dryer at-60 deg.C until the water content is below 14% to obtain natural high molecular fish skin collagen for medical grade biomaterial.
Example 2
Taking 200g of fresh tilapia skin to remove scales, residual meat and fat, cutting into small blocks of 1cm multiplied by 1cm by a slicer, rinsing with 20 times of pure water at 4 ℃ for 30 minutes, repeating the steps once, and draining off water. Putting the fish skin into 4% NaCl solution at 4 ℃ with the material-liquid ratio of 1:30 (w/w), stirring for 36 hours, changing the solution every 8 hours, draining water, washing with deionized water at 4 ℃ for 1:20 (w/w) for 4 times, 30min each time, and draining water.
Putting the fish skin into 0.1M NaOH solution at 4 ℃, stirring for 48 hours according to the material-liquid ratio of 1:30 (w/w), changing the solution every 12 hours, and draining water. Washing with deionized water at 4 deg.C (1: 30 w/w) for 3 times (1 hr each time) until Ph is 7-8, and draining off water.
Putting the cleaned fish skin into 0.5M acetic acid solution at 4 deg.C, mashing with tissue triturator at a ratio of 1:30, stirring for 4 hr, centrifuging at 4 deg.C with 10000rpm, and collecting supernatant. The precipitate was put into 0.5M acetic acid solution at 4 ℃ in a ratio of 1:20, stirred for 4 hours, centrifuged at 10000rpm at 4 ℃ and the supernatant was collected. The supernatants were combined 2 times, and 0.3% pepsin was added to the supernatants, and the mixture was treated with continuous stirring at 4 ℃ for 48 hours.
Adding saturated NaCl solution to salt out to make NaCl concentration in the feed liquid be 4%, precipitating and standing. Centrifuging the salting-out solution at 4 deg.C at 10000rpm, collecting precipitated crude collagen, dissolving crude collagen in pure water at 4 deg.C and 0.5M at a ratio of 1:10 (w/w), dialyzing with pure water at 1:20 (w/w) and semipermeable membrane with molecular weight cutoff of 100KD for 48 hr, and replacing the dialyzed solution every 8 hr to obtain purified collagen solution.
And (3) putting the purified collagen liquid into a low-temperature freeze dryer at the temperature of-60 ℃ for freeze-drying until the water content is below 14 percent, thereby obtaining the natural high-molecular fish skin collagen for the medical-grade biomaterial.
FIG. 2 shows an SDS-PAGE pattern of fish skin collagen according to the invention. The map adopts the fish skin collagen of the fresh tilapia in the embodiment. The numbers below the symbol M are the protein molecular weight markers in Kilodaltons (KD). Symbol a is Bovine Serum Albumin (BSA), symbol B is Marker (Marker), and symbol C is fish skin collagen. Lane 1 is a hetero protein, lane 2 is a trimer, lane 3 is a dimer, lane 4 is α1The strip 5 being alpha2. As can be seen from fig. 2, the method for preparing fish skin collagen according to the present invention can obtain highly pure collagen, and the specific comparative data are shown in table 1:
table 1: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) map analysis of fish skin collagen of fresh tilapia
| Strip number | Band content (%) | Molecular weight (KD) | Relative mobility |
| 1 | 0.81 | 372.73 | 0.028 |
| 2 | 2.28 | 328.79 | 0.072 |
| 3 | 48.09 | 257.58 | 0.142 |
| 4 | 34.11 | 116.90 | 0.313 |
| 5 | 14.71 | 108.44 | 0.342 |
By adopting the preparation method of the fish skin collagen, the content of the fish skin collagen of the fresh tilapia can reach 99.19 percent.
Through enzyme-linked immunoassay of rat type I collagen (col-I), the enzyme-soluble fish skin collagen prepared by the method has biocompatibility greatly superior to that of acid-soluble fish skin collagen, and completely meets the low antigenicity requirement of biomedical materials, and specific comparative analysis is shown in Table 2, wherein the fish skin collagen of the fresh tilapia mossambica in the embodiment is adopted in the Table 2.
Table 2: enzyme-linked immunoassay of rat type I collagen (col-I)
| Material | Type I collagen content in rat serum |
| Acid soluble fish skin collagen | 30.182μg/L |
| Enzyme soluble fish skin collagen (method) | 16.150μg/L |
By adopting the preparation method of the fish skin collagen for the medical grade biomaterial, the non-collagen components of the fish skin can be sufficiently and effectively removed, the high-purity collagen with the hydroxyproline content of more than or equal to 9 percent, the non-collagen content of less than or equal to 1 percent and the ash content of less than or equal to 1 percent is obtained, the fish resources can be utilized in a larger range, the process is simple and easy to operate, the processing time is short, the preparation method is suitable for industrial production, and the biocompatibility effect is obvious.
The foregoing describes specific embodiments of the present invention. However, those skilled in the art will appreciate that various modifications and substitutions can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention. Such modifications and substitutions are intended to be included within the scope of the present invention as defined by the appended claims.
Claims (10)
1. A preparation method of fish skin collagen for medical grade biomaterial is characterized in that the fish skin collagen is prepared according to the following steps:
1) raw material treatment: mincing the skin of the cleaned and dehydrated marine fish or freshwater fish into 0.5-5 cm2Adding 2-5% NaCl salt solution with the material-liquid ratio of 1:30 (w/w), stirring for 8-12 hours at 0-4 ℃, removing waste liquid, repeating for 3-4 times, cleaning with deionized water, putting the cleaned fish skin into 0.05-0.2M NaOH solution with the material-liquid ratio of 1:30 (w/w), stirring for 32-48 hours at intervals of 8-12 hoursReplacing the NaOH solution once, and then fully cleaning the fish skin treated by the alkali solution by using deionized water until the fish skin is neutral;
2) extracting collagen: putting the cleaned fish skin into 0.2-1M acid solution according to the material-liquid ratio of 1: 10-30 (w/w), mashing, stirring for 4-8 hours, centrifuging, taking supernatant to obtain acid-soluble collagen solution, repeating for 2-3 times, combining the centrifuged supernatant, adding 0.1-1% of protease, stirring for 24-60 hours, and performing enzymatic treatment to obtain enzymatic hydrolysis collagen solution;
3) and (3) purifying an extract: adding saturated NaCl solution until the salt content in the collagen liquid reaches 2-5%, standing for 2-6 hours, centrifuging to obtain precipitated crude collagen, dissolving the crude collagen in pure water according to the material-liquid ratio of 1: 5-10 (w/w), dialyzing for 4-6 times by using a semipermeable membrane, purifying the collagen liquid, and carrying out freeze drying treatment at-60 ℃ to obtain the natural high-molecular fish skin collagen for the medical-grade biomaterial, wherein the water content of the natural high-molecular fish skin collagen is less than or equal to 14%.
2. The method for preparing fish skin collagen according to claim 1, wherein the fish skin used in step 1) is fresh or frozen sea fish or freshwater fish skin.
3. The method for preparing fish skin collagen according to claim 2, wherein the sea fish skin is cod skin, sole skin, squid skin, and salmon skin, and the fresh water skin is tilapia skin, black carp skin, grass carp skin, silver carp skin, and bighead carp skin.
4. The method for preparing fish skin collagen according to claim 1, wherein the step of washing the fish skin in step 1) comprises the step of removing impurities such as fish scales, meat residues and fat from the fish skin of fresh or frozen marine fish or freshwater fish in a feed-to-liquid ratio of 1: stirring with 20-30 (w/w) 0-4 ℃ pure water, cleaning for 30 minutes, removing waste water, and repeating once.
5. The method for preparing fish skin collagen according to claim 1, wherein the step of treating the fish skin to be neutral in step 1) comprises: rinsing with deionized water for 4 times, wherein each time lasts for 0.5-1 hour, and the pH is 7-8.
6. The method for preparing fish skin collagen according to claim 1 or 5, wherein the temperature of the deionized water in the step 1) is 0-4 ℃, and the ratio of the fish skin to the deionized water is 1:30 (w/w).
7. The method for preparing fish skin collagen according to claim 1, wherein the acidic solution in step 2) is an acetic acid, phosphoric acid or citric acid solution.
8. The method for preparing fish skin collagen according to claim 1, wherein the protease in step 2) is pepsin, trypsin or alkaline protease.
9. The method for preparing fish skin collagen according to claim 1, wherein the step of dialyzing the semipermeable membrane in the step 3) comprises: and (3) using a semipermeable membrane with the molecular weight cutoff of 100KD, wherein the dialyzed external liquid is pure water with the material-liquid ratio of 1: 20-40 (w/w) at 0-4 ℃, the dialyzed liquid is replaced once every 8-12 hours, and the process is repeated for 4-6 times.
10. The method for preparing fish skin collagen according to claim 1, wherein the step of preparing fish skin collagen is performed at 0-4 ℃.
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