CN111302319A - Method for extracting hydroxyapatite and collagen from fish scales by using eutectic solvent - Google Patents
Method for extracting hydroxyapatite and collagen from fish scales by using eutectic solvent Download PDFInfo
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- CN111302319A CN111302319A CN201811507736.2A CN201811507736A CN111302319A CN 111302319 A CN111302319 A CN 111302319A CN 201811507736 A CN201811507736 A CN 201811507736A CN 111302319 A CN111302319 A CN 111302319A
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- hydroxyapatite
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- 229910052588 hydroxylapatite Inorganic materials 0.000 title claims abstract description 65
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 title claims abstract description 65
- 102000008186 Collagen Human genes 0.000 title claims abstract description 55
- 108010035532 Collagen Proteins 0.000 title claims abstract description 55
- 229920001436 collagen Polymers 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 48
- 241000251468 Actinopterygii Species 0.000 title claims abstract description 47
- 239000002904 solvent Substances 0.000 title claims abstract description 32
- 230000005496 eutectics Effects 0.000 title claims abstract description 31
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 12
- 239000002244 precipitate Substances 0.000 claims abstract description 11
- 239000000843 powder Substances 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 8
- 238000005119 centrifugation Methods 0.000 claims abstract description 7
- 239000012716 precipitator Substances 0.000 claims abstract description 6
- 238000002390 rotary evaporation Methods 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 229910052739 hydrogen Inorganic materials 0.000 claims description 8
- 239000001257 hydrogen Substances 0.000 claims description 8
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims description 7
- 235000019743 Choline chloride Nutrition 0.000 claims description 7
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical group [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims description 7
- 229960003178 choline chloride Drugs 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 3
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- 238000000746 purification Methods 0.000 claims 2
- 150000001298 alcohols Chemical group 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 14
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 abstract description 6
- 238000003912 environmental pollution Methods 0.000 abstract description 5
- 239000002608 ionic liquid Substances 0.000 abstract description 5
- 238000003786 synthesis reaction Methods 0.000 abstract description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- 150000001413 amino acids Chemical class 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000003837 high-temperature calcination Methods 0.000 abstract description 3
- 230000009467 reduction Effects 0.000 abstract description 2
- 238000010521 absorption reaction Methods 0.000 description 24
- 102000004169 proteins and genes Human genes 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 16
- 150000001408 amides Chemical class 0.000 description 7
- 238000005452 bending Methods 0.000 description 7
- 229910019142 PO4 Inorganic materials 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 239000013078 crystal Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 210000000988 bone and bone Anatomy 0.000 description 4
- 238000002329 infrared spectrum Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 102000012422 Collagen Type I Human genes 0.000 description 3
- 108010022452 Collagen Type I Proteins 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
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- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- ZIBGPFATKBEMQZ-UHFFFAOYSA-N triethylene glycol Chemical compound OCCOCCOCCO ZIBGPFATKBEMQZ-UHFFFAOYSA-N 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 238000002441 X-ray diffraction Methods 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 2
- 159000000007 calcium salts Chemical class 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 229910001385 heavy metal Inorganic materials 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000007873 sieving Methods 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- 241000242757 Anthozoa Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 235000014653 Carica parviflora Nutrition 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000010796 biological waste Substances 0.000 description 1
- 238000001354 calcination Methods 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003278 egg shell Anatomy 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000000374 eutectic mixture Substances 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 239000011094 fiberboard Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 231100000378 teratogenic Toxicity 0.000 description 1
- 230000003390 teratogenic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B25/00—Phosphorus; Compounds thereof
- C01B25/16—Oxyacids of phosphorus; Salts thereof
- C01B25/26—Phosphates
- C01B25/32—Phosphates of magnesium, calcium, strontium, or barium
-
- 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]
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/70—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data
- C01P2002/72—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data by d-values or two theta-values, e.g. as X-ray diagram
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/80—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
- C01P2002/82—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70 by IR- or Raman-data
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/80—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
- C01P2002/84—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70 by UV- or VIS- data
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- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Inorganic Chemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
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- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
A method for extracting hydroxyapatite and collagen from fish scales by using a eutectic solvent. Reacting the treated fish scale powder and the eutectic solvent at a mass ratio of 1:5-25 at 25-100 ℃ for 2-5h, and centrifuging. And purifying the precipitate by 5 percent sodium hydroxide solution, and drying to obtain the hydroxyapatite. Adding a precipitator into the supernatant for centrifugation, purifying the precipitate with an acetic acid solution to obtain regenerated collagen, and recovering the eutectic solvent from the supernatant through rotary evaporation for reuse. The method adopts the eutectic solvent, is simple to operate, low in cost, green, safe and pollution-free. The problems of safety and the reduction of the bioactivity of the hydroxyapatite caused by a high-temperature calcination method in the chemical synthesis of the hydroxyapatite are solved; the problems of damaged amino acid, high cost and environmental pollution in the collagen extraction by the acid-base enzyme method are solved. The problem of high price of the imidazole ionic liquid is avoided. The hydroxyapatite and the collagen extracted by the method have high purity, and a new method for efficiently utilizing resources is provided, so that the method is beneficial to sustainable development.
Description
Technical Field
The invention relates to a method for extracting hydroxyapatite and collagen from fish scales by using a eutectic solvent, belonging to the technical field of extraction processes.
Background
In the process of processing and producing fish in China, tens of thousands of tons of fish scales are produced. The fish scale structure can be divided into two layers: the upper layer is mainly made of inorganic components such as hydroxyapatite and the like, and the lower layer is mainly made of a fiber board layer of protein. The fish scales mainly contain hydroxyapatite and collagen. The substances are extracted from the fish scales, so that the added value of the fish can be increased, and the environmental pollution can be reduced. Hydroxyapatite and collagen can also be used in many fields, such as the food field, the biomedical field, the electrochemical field and the cosmetic field. The extracted collagen can be made into edible protein film, sheet or powder collagen hemostatic, or collagen facial mask. The hydroxyapatite in the fish scales can be used as an adsorption material of heavy metals and dyes, a drug carrier, a bone repair replacement material and the like. The extraction of hydroxyapatite and collagen in fish scales can not only improve the economic value of fish bodies, promote the development of aquatic product processing industry, but also reduce environmental pollution, thereby creating good economic and social benefits and being beneficial to sustainable development.
The preparation method of the hydroxyapatite comprises a chemical synthesis method and a natural hydroxyapatite preparation method. The preparation method of the natural hydroxyapatite comprises a high-temperature calcination method and a solvent extraction method. The solvent extraction method includes an acid method, an alkaline method, an enzymatic method, an acid-enzyme complex method, and the like. The chemical synthesis of hydroxyapatite has the problems of high reaction temperature, long reaction time, small product activity, wide size distribution range, non-uniformity, environmental pollution, poor safety and the like. Therefore, in recent years, researchers have begun to prepare natural hydroxyapatite from various biological waste materials, such as eggshells, corals, pig bones, bovine bones, fish scales, and the like. Compared with chemical synthesis, the latter has the advantages of high biological safety, low cost and the like. In addition, the natural hydroxyapatite contained in the hydroxyapatite is abundant in resources and low in price, so that extraction of the hydroxyapatite from natural substances becomes a hot point of research in recent years. However, the preparation of hydroxyapatite by calcination at extremely high temperature causes the decomposition and falling of hydroxyl groups in HA crystal lattices to form anoxic hydroxyapatite, and the biological activity is also reduced; the cost of enzymatic extraction is high; in the acid-base extraction method, the use of a large amount of acid and base can damage instruments and equipment. And also pollutes the environment. Therefore, the method for extracting the hydroxyapatite by adopting the new method has great development prospect. The extraction method of collagen comprises alkaline method, salt method, acid method, hot water method, enzyme method and ionic liquid extraction method. The hot water extraction method is characterized in that the collagen peptide chain is decomposed into a single chain form from a right-handed helix during heating, and the single chain form is slightly denatured. Acid extraction, rapid and complete, but destroys some serine and tyrosine. And acid can not be adopted during pretreatment, otherwise, the foreign protein is difficult to remove. The alkali extraction method is simple, convenient, rapid and thorough in operation, but can destroy some amino acids such as hydroxyproline and strong lysine. In addition, racemization may occur, which may cause extracted collagen to be teratogenic and carcinogenic. In addition, the collagen extracted by the method has small relative molecular weight and low application value. The temperature for extracting collagen by the enzyme method is low, the destructiveness to collagen molecules can be controlled, but the required time is long. The increase in temperature will destroy the collagen triple helix structure to some extent and will reduce its thermal stability. In addition, enzymatic extraction is costly. The eutectic solvent can be used as a direct solvent of the collagen, the structure of the collagen can be maintained, and the structure of the collagen is not changed before and after the collagen is dissolved and regenerated, so that the good biological activity of the collagen is maintained.
The eutectic solvent is a eutectic mixture formed by two or three components in proportion through the interaction of hydrogen bonds. The ionic liquid has the similar properties to the traditional ionic liquid, and has the advantages of low raw material price, simple preparation, safety, no toxicity or low toxicity. The anions and cations of the eutectic solvent can destroy hydrogen bonds between and in the peptide chains of the protein and form stronger hydrogen bonds with the peptide chains, so that the protein is dissolved in the eutectic solvent. Dissolving the protein, adding a precipitator to regenerate the protein, and purifying to obtain the collagen. After the protein is dissolved, other substances are purified to obtain the hydroxyapatite. Finally, the eutectic solvent is recovered by removing the precipitant by rotary evaporation.
Disclosure of Invention
The invention aims to provide a method for extracting hydroxyapatite and collagen from fish scales by using a eutectic solvent. The method has simple operation and low cost, and can comprehensively extract hydroxyapatite and collagen. Meanwhile, the problems of safety and the reduction of the bioactivity of the hydroxyapatite caused by a high-temperature calcination method in the chemical synthesis of the hydroxyapatite are avoided; the problems of damaged amino acid, high cost and environmental pollution in the collagen extraction by the acid-base enzyme method are solved. The problem of high price of the imidazole ionic liquid is avoided. The hydroxyapatite and the collagen extracted by the method have high purity, and a new method for efficiently utilizing resources is provided, so that the method is beneficial to sustainable development.
The technical scheme of the invention is as follows:
1) the method comprises the steps of washing fresh crucian scales collected from farmer markets by tap water, and repeatedly washing by distilled water. And (5) naturally drying the cleaned fish scales or drying the fish scales in an oven (only a fan is started). Pulverizing dried fish scales, sieving with 80-150 mesh sieve, transferring into container, sealing with sealing film, and storing in drier.
2) The scale powder and the eutectic solvent are heated and stirred in a round-bottom flask for extraction reaction at a temperature of 25-100 ℃ for 2-5h according to a ratio of 1: 5-25.
3) After the reaction is finished, centrifuging, repeatedly washing the precipitate with distilled water, and removing the eutectic solvent.
4) And (3) reacting the fish scales for 5 hours at 70 ℃ by using a 5% sodium hydroxide solution to remove grease in the fish scales. Centrifuging, washing the precipitate with deionized water repeatedly to remove sodium hydroxide, and drying at 70-105 deg.C to obtain hydroxyapatite.
5) Adding a precipitator into the centrifuged supernatant in the step 2, wherein the volume ratio of the precipitator to the eutectic solvent is 5-15:1, and centrifuging after precipitating for 12-24 h. The precipitate was repeatedly washed with a precipitant, and then 0.5mol/L acetic acid solution was added thereto, and dialyzed against distilled water at 4 ℃.
6) Centrifuging, and freeze-drying the supernatant for 24-48h to obtain the collagen.
7) And 3, taking the centrifuged supernatant in the step 3 as a mixture of the eutectic solvent and the precipitant, removing the precipitant through rotary evaporation, and recovering to obtain the eutectic solvent.
8) The eutectic solvent can be choline chloride/glycerol, choline chloride/1, 4-butanediol, choline chloride/triethylene glycol, choline chloride/ethylene glycol, etc.
9) The precipitant used can be ethanol and deionized water.
Compared with the prior art, the invention has the following advantages:
the used eutectic solvent is simple in synthetic method and low in raw material price; the extraction process is simple and pollution-free, and the problems of corrosion to equipment, pollution to the environment and high cost in the traditional method are avoided; the eutectic solvent can be recycled.
Drawings
FIG. 1 is a Fourier infrared spectrum of fish scale powder and extracted hydroxyapatite
FIG. 2 is an X-ray diffraction analysis chart of extracted hydroxyapatite
FIG. 3 is a Fourier infrared spectrum of extracted collagen
FIG. 4 is a UV analysis chart of extracted collagen
Detailed Description
The present invention is further described in detail by the following examples, but the technical content described in the examples is illustrative and not restrictive, and the scope of the present invention should not be limited thereby.
Example one
1) Fresh crucian scales collected from farmer markets are washed by tap water to remove impurities such as dust, fish silver, mucin and the like on the surfaces of the scales. Repeated washings with deionized water were performed to remove mineral and chloride impurities from the tap water. And (5) naturally drying the cleaned fish scales or drying the fish scales in an oven (only a fan is started). Crushing the dried fish scales, sieving the crushed fish scales with a 80-mesh sieve, transferring the crushed fish scales into a container, sealing the container with a sealing film, and storing the sealed fish scales in a dryer for later use.
2) 3g of fish scale powder and 6g of choline chloride/triethylene glycol were added to a round bottom flask and stirred magnetically at 80 ℃ for 2 h. After completion, the liquid in the round bottom flask was transferred to a centrifuge tube for centrifugation.
3) The precipitate is washed clean with distilled water (which can be identified with silver nitrate solution until no white precipitate is formed) to obtain a coarse extract of hydroxyapatite. Dissolving with 5% sodium hydroxide at a ratio of 1:7 at 70 deg.C under stirring for 5 hr, washing with deionized water to neutrality, and drying to obtain pure HA.
4) The supernatant is a mixture of protein and eutectic solvent, 40mL of ethanol precipitator is added, and centrifugation is carried out after 24h of precipitation.
5) After centrifugation, the precipitate was washed with absolute ethanol to obtain a crude collagen extract, which was dissolved in 0.5mol/L acetic acid solution and transferred to a dialysis bag, followed by dialysis with deionized water in a freezer at 4 ℃. Changing water 3-4 times every day until the dialysate is neutral. Transferring the solution in the dialysis bag to a culture dish, pre-freezing, and vacuum freeze-drying for 24h to obtain pure collagen.
6) And 5) rotatably evaporating the centrifuged supernatant at 80 ℃ to remove ethanol to obtain choline chloride/triethylene glycol.
Explanation of the drawings
FIG. 1 is an infrared spectrum of fish scale powder and extracted hydroxyapatite. The fish scale powder is 1546.23cm-1、1241.70cm-1The absorption peaks appeared in the sample correspond to the absorption peaks of amide II and amide III, and the absorption peaks do not exist in the extracted hydroxyapatite. At 3431.77cm-1The absorption peak appeared corresponding to the stretching vibration peak of-OH. The absorption peak appearing at 1636.99 is the bending vibration peak of-OH. Appearing at 1455.38cm-1And 1419.62cm-1Absorption peak at (B) corresponds to CO3 2-The asymmetric stretching vibration peak of (1). Appearing at 873.86cm-1Absorption peak at (B) corresponds to CO3 2-The bending vibration peak of (1). Description of CO3 2-Enters the inside of the hydroxyapatite crystal. By substitution of a part of OH-And PO4 3-AB mixed type carbonate hydroxyapatite. 1419.62cm-1And 873.86cm-1A strong absorption peak appears at, and represents CO3 2-To pair-PO4 3-The substitute of (2) is B-type hydroxyapatite. 1560cm-1An extremely weak absorption peak is present, and is CO3 2-To OH-The substituted compound is A-type hydroxyapatite. 1455.38cm-1The absorption peak appeared to be CO3 2-Combined substitution of OH-And PO4 3-The absorption peak of (1). The extracted hydroxyapatite is mainly B-type hydroxyapatite. At 1038.63cm-1、603.22cm-1、565.61cm-1The absorption peak appeared at corresponds to PO4 3-The stretching vibration peak, the symmetric bending vibration peak and the asymmetric bending vibration peak. At 603.50cm-1And 565.61cm-1A sharp vibration peak appears, which indicates that the extracted hydroxyapatite has a good crystal structure. The Fourier infrared spectrogram of the hydroxyapatite does not have a characteristic absorption peak of protein, and the extracted hydroxyapatite is proved to be free of protein. The spectrogram contains CO3 2-And PO4 3-The absorption peak of (a) proves that the extracted HA is a mixed calcium salt of carbonate type and phosphate type. The extracted mixed calcium salt hydroxyapatite is closer to the inorganic component of human hard tissue, and can be better applied to the fields of bone repair substitute materials and the like. At the same time, CO3 2-The solubility of the hydroxyapatite and the adsorption of heavy metal ions are increased.
Fig. 2 is an X-ray diffraction analysis diagram of hydroxyapatite on the market, wherein the main intensity peak of the hydroxyapatite is at 32.13 degrees, the secondary intensity peak is at 25.91 degrees, and the crystal face is at 49.46 degrees. The main intensity peak and the secondary intensity peak of the extracted hydroxyapatite are respectively at 32.08 degrees and 25.94 degrees, and the crystal face is at 49.45 degrees, which is basically consistent with the hydroxyapatite sold in the market. No obvious impurity peak, which shows that the purity of the extracted hydroxyapatite is higher.
Fig. 3 is a graph of fourier infrared analysis of extracted collagen. At 3423.65cm-1An absorption peak appears, which corresponds to the stretching vibration peak of N-H in the amide A band, and the peak is moved to low frequency by hydrogen bond association. At 3096.63cm-1And 2959.37cm-1The absorption peaks appeared at the positions correspond to the unsaturated stretching vibration peak and the saturated stretching vibration peak of C-H in the amide B band respectively. 1654.41cm-1The absorption peak at the position corresponds to the C ═ O stretching vibration peak of the amide I band and corresponds to the secondary structure- α helix of the protein, because the C atom is connected with N-H, the characteristic of C ═ O double bond is reduced, the bond force constant is reduced, the vibration frequency moves to low frequency, 1650--1In the random coil, no absorption peak appears in this range. 1546.19cm-1The absorption peak appeared here corresponds to the N-H bending vibration peak of the amide II band. 1454cm-1The absorption peak appeared at corresponds to-CH2The bending vibration peak of (1). 1239.72cm-1The peak of C-N bending vibration corresponding to the amide III band corresponds to β folding of protein, the structure of the protein is consistent with that of type I collagen reported in the literature, and the length of the protein is 1240cm-1And 1450cm-1The ratio of absorbance was 1.02, between 1 and 1.2, and the ratio dropped to 0.6 when the structure was completely destroyed, indicating that the collagen was not denatured. In addition, the characteristic absorption peak of HA does not appear in the fourier infrared spectrum of the extracted collagen, indicating that the purity of the extracted collagen is high. The extracted collagen has good purity and integrity.
Fig. 4 is a uv analysis chart of extracted collagen. A strong absorption peak at 230nm was observed, which is consistent with the characteristics of type I collagen. No absorption peak in the range of 250-400nm, no absorption peak of conjugated double bond in tyrosine or tryptophan, and no other proteins such as keratin. The extracted collagen is typical type I collagen and has higher purity.
Claims (10)
1. A method for extracting hydroxyapatite and collagen in fish scales by using a eutectic solvent is characterized in that the eutectic solvent is used for extracting the hydroxyapatite and the collagen in the fish scales, and the process flow is as follows: adding the fish scale powder into a eutectic solvent according to a certain proportion, reacting for a period of time at a certain temperature, centrifuging to respectively obtain a precipitate and a supernatant, purifying the precipitate, and drying to obtain hydroxyapatite; adding a precipitator into the supernatant to obtain collagen, and performing rotary evaporation on the supernatant to recover the eutectic solvent.
2. The method for extracting hydroxyapatite and collagen from fish scales according to claim 1, wherein the collected fish scales are washed, dried, crushed and sieved.
3. The method for extracting hydroxyapatite and collagen from fish scales according to claim 1, wherein the eutectic solvent is a mixture of binary or ternary components formed by a hydrogen bond donor and a hydrogen bond acceptor according to a certain ratio, the quaternary ammonium salt hydrogen bond acceptor is choline chloride, and the hydrogen bond acceptor is alcohols.
4. The method for extracting hydroxyapatite and collagen from fish scales by using the eutectic solvent as claimed in claim 1, wherein the mass ratio of the fish scale powder to the eutectic solvent is 1:5-25, the temperature is 25-100 ℃ and the time is 2-5h, and the fish scale powder and the eutectic solvent are heated and stirred in a round-bottomed flask.
5. The method for extracting hydroxyapatite and collagen from fish scales according to claim 1, wherein the centrifugation condition is 5000-.
6. The method for extracting hydroxyapatite and collagen from fish scales according to claim 1, wherein the precipitate after centrifugation according to the conditions of claim 5 is repeatedly washed with distilled water to remove the eutectic solvent, and is heated at 70 ℃ for 5 hours under stirring at a ratio of 1:3-9 with sodium hydroxide, and then repeatedly washed with distilled water to remove sodium hydroxide and dried to obtain hydroxyapatite.
7. The method for extracting hydroxyapatite and collagen from fish scales according to claim 1, wherein a precipitant is added to the supernatant, and the collagen is obtained after centrifugation, washing, purification and freeze-drying.
8. The method for extracting hydroxyapatite and collagen from fish scales according to claim 7, wherein the volume ratio of the precipitant to the supernatant is 9-11: 1.
9. The method for extracting hydroxyapatite and collagen from fish scales according to claim 7, wherein the precipitating agent is: ethanol and deionized water.
10. The method for extracting hydroxyapatite and collagen from fish scales according to claim 7, wherein the selected reagent for purification is 0.5mol/L acetic acid solution, and the freeze-drying time is 24-48h after dialysis at 4 ℃.
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