CN110818791A - Efficient preparation method of fish collagen - Google Patents
Efficient preparation method of fish collagen Download PDFInfo
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- CN110818791A CN110818791A CN201911151753.1A CN201911151753A CN110818791A CN 110818791 A CN110818791 A CN 110818791A CN 201911151753 A CN201911151753 A CN 201911151753A CN 110818791 A CN110818791 A CN 110818791A
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- 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, cold insoluble globulin [CIG]
Abstract
The invention relates to a preparation method of fish collagen, which comprises the following steps: step S1, fish scale pretreatment, step S2, microwave-assisted ultrasonic extraction, and step S3, purification and post-treatment. The invention adopts a preparation method combining double-frequency ultrasound and double-frequency microwave-assisted extraction, the method combines the advantages of various fish collagen preparation methods in the prior art, the fish collagen has higher yield, better purity and lower preparation cost, the raw material used in the process has wide sources, the preparation process is simple and easy to operate, the preparation method is suitable for large-scale production, and the prepared collagen product has high quality and high yield; before extraction, degreasing cleaning liquids with different properties are adopted for cleaning, the cleaning effect is better, the dissolution of fat is facilitated, the later-stage extraction is promoted, and the prepared fish collagen is higher in purity.
Description
Technical Field
The invention relates to the technical field of biological products, in particular to an efficient preparation method of fish collagen.
Background
Collagen is a natural protein synthesized by fibroblasts in an animal body, is a structural protein of extracellular matrix, is also called collagen, and widely exists in connective tissues such as bones, skins, tendons and the like of animals, so that tissues and organs have good mechanical structures, thereby playing roles in supporting the organs and protecting the organism. The collagen and its degradation products are rich in glycine, serine, alanine, aspartic acid and other natural moisturizing factors and a large amount of hydrophilic groups, so that it has a moisturizing function. In addition, collagen has good permeability, can be filled between skin matrixes and absorbed by the skin, and has the effects of repairing the skin and whitening the skin, so that collagen and collagen peptide which is a degradation product of the collagen are more and more widely applied to beauty care and cosmetics.
The fish collagen is collagen produced by taking fish as a raw material, so that the quality of the collagen is improved, the source of the collagen raw material is enlarged, waste is changed into valuable, and resources are saved. The existing methods for producing fish collagen mainly comprise a hot water extraction method, a salt method, an acid method, an alkaline method, an enzyme method and the like. The reaction condition for producing collagen by the enzyme method is mild, the triple helix structure of the collagen is not damaged, and the method can keep the characteristics and is widely applied. However, the existing enzymolysis technology is carried out at normal temperature and normal pressure or even at high temperature and high pressure, the enzymolysis, concentration and drying processes all need higher temperature, the influence on the activity of the fish collagen is larger, the product quality is difficult to ensure, the powder yield is lower, and the small-batch production cannot be realized. In addition, the existing production methods also have the defects of higher production cost, poor quality of finished products, longer production period of products, larger volume of extracting solution, incomplete impurity removal of enzymatic hydrolysate and larger enzyme dosage.
Therefore, how to efficiently prepare high-quality collagen is a technical problem to be solved by those skilled in the art.
Disclosure of Invention
The invention aims to provide a high-efficiency preparation method of fish collagen for overcoming the defects of the prior art; the preparation method adopts a preparation method combining double-frequency ultrasound and double-frequency microwave-assisted extraction, combines the advantages of various fish collagen preparation methods in the prior art, has higher yield and purity of fish collagen, lower preparation cost, wide raw material sources used in the process, simple and easy operation of the preparation process, suitability for large-scale production, and high quality and yield of the prepared collagen product; before extraction, degreasing cleaning liquids with different properties are adopted for cleaning, the cleaning effect is better, the dissolution of fat is facilitated, the later-stage extraction is promoted, and the prepared fish collagen is higher in purity.
In order to achieve the purpose, the invention adopts the technical scheme that:
step S1, fish scale pretreatment: adding fish phosphorus into water containing acetic acid and salt, soaking and softening for 2-3 hours at 35-45 ℃, then adding an acidic degreasing cleaning solution for rinsing for 20-30 minutes, then adding an alkaline degreasing cleaning solution for rinsing for 15-25 minutes, then rinsing with distilled water to neutrality, then finely cutting fish scales, washing with ethanol for 3-5 times, putting into a stirrer for stirring, washing with 0.03-0.06mol/L Tris-HCl buffer solution containing 4-6mol/L NaCl for 1-2 hours, and finally centrifuging to take precipitates;
step S2, extraction: adding the fish scales prepared in the step S1 into the extracting solution, carrying out shearing extraction at 30-60 ℃ to enable the grain size of the fish scales to reach 10-100 mu m, then carrying out rotary evaporation to remove the extracting solution to obtain a crude extract, adding a sodium chloride solution with the mass fraction of 0.3-0.5% and 0.5mol/L of glacial acetic acid into the crude extract, stirring for 1-2 hours, then adding a decolorizing agent to carry out decolorization and deodorization, and taking supernatant to obtain an extraction stock solution;
step S3, purification and post-treatment: slowly adding 4-6mol/L sodium carbonate into the extraction stock solution prepared in the step S2 to adjust the pH value to 7, centrifuging to remove precipitates, then filling the rest solution into a dialysis bag, respectively dialyzing for 1-2d by using acetic acid solutions with the mass fractions of 0.1% and 0.01%, dialyzing for 2-3d by using distilled water, changing the dialyzate twice every day, and finally obtaining the fish collagen aqueous solution with higher purity; the purified collagen aqueous solution is prepared into a pure fish collagen sample by adopting a low-temperature freeze drying method.
Further, in step S1, the mass ratio of the fish phosphorus, the white vinegar, the salt, the water, the acidic degreasing cleaning solution and the alkaline degreasing cleaning solution is 1 (0.2-0.4): (0.1-0.3): 10-20): 8-10): 10-12.
Further, the acidic degreasing cleaning solution is prepared from the following raw materials in parts by weight: 5-10 parts of glycyrrhizic acid, 10-15 parts of citric acid, 3-5 parts of ethylenediamine tetraacetate, 0.3-0.7 part of coconut diethanolamide and 120 parts of water 100-.
Further, the alkaline degreasing cleaning solution is prepared from the following raw materials in parts by weight: 10-15 parts of nicotine, 20-30 parts of dipropylene glycol, 3-5 parts of sodium oleate, 0.3-0.7 part of cocamidopropyl betaine and 120 parts of water.
Further, in the step S2, the mass ratio of the fish scales to the extracting solution is 1 (20-30).
Further, the mass ratio of the crude extract, the sodium chloride solution and the glacial acetic acid in the step S2 is 1 (20-30) to 10-15.
Preferably, the extracting solution is at least one of water, ethanol and ethyl acetate.
Further, the decolorizing agent is selected from at least one of activated carbon, fly ash and chitosan.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages: the preparation method combines the advantages of various fish collagen preparation methods in the prior art, the fish collagen has higher yield, better purity and lower preparation cost, the raw materials used in the process are wide in source, the preparation process is simple and easy to operate, the preparation method is suitable for large-scale production, and the prepared collagen product has high quality and high yield; before extraction, degreasing cleaning solutions with different properties are adopted for cleaning, the cleaning effect is better, lipid dissolution is facilitated, and the later extraction is promoted, so that the prepared fish collagen has higher purity, the preparation process is safe, sanitary and harmless, and the social value and the economic value are higher.
Detailed Description
The invention relates to step S1, fish scale pretreatment: adding fish phosphorus into water containing acetic acid and salt, soaking and softening for 2-3 hours at 35-45 ℃, then adding an acidic degreasing cleaning solution for rinsing for 20-30 minutes, then adding an alkaline degreasing cleaning solution for rinsing for 15-25 minutes, then rinsing with distilled water to neutrality, then finely cutting fish scales, washing with ethanol for 3-5 times, putting into a stirrer for stirring, washing with 0.03-0.06mol/L Tris-HCl buffer solution containing 4-6mol/L NaCl for 1-2 hours, and finally centrifuging to take precipitates;
step S2, extraction: adding the fish scales prepared in the step S1 into the extracting solution, carrying out shearing extraction at 30-60 ℃ to enable the grain size of the fish scales to reach 10-100 mu m, then carrying out rotary evaporation to remove the extracting solution to obtain a crude extract, adding a sodium chloride solution with the mass fraction of 0.3-0.5% and 0.5mol/L of glacial acetic acid into the crude extract, stirring for 1-2 hours, then adding a decolorizing agent to carry out decolorization and deodorization, and taking supernatant to obtain an extraction stock solution;
step S3, purification and post-treatment: slowly adding 4-6mol/L sodium carbonate into the extraction stock solution prepared in the step S2 to adjust the pH value to 7, centrifuging to remove precipitates, then filling the rest solution into a dialysis bag, respectively dialyzing for 1-2d by using acetic acid solutions with the mass fractions of 0.1% and 0.01%, dialyzing for 2-3d by using distilled water, changing the dialyzate twice every day, and finally obtaining the fish collagen aqueous solution with higher purity; the purified collagen aqueous solution is prepared into a pure fish collagen sample by adopting a low-temperature freeze drying method.
Further, in the step S1, the mass ratio of the fish phosphorus, the white vinegar, the salt, the water, the acidic degreasing cleaning solution and the alkaline degreasing cleaning solution is 1 (0.2-0.4): 0.1-0.3): 10-20): 8-10): 10-12; the acidic degreasing cleaning solution is prepared from the following raw materials in parts by weight: 5-10 parts of glycyrrhizic acid, 10-15 parts of citric acid, 3-5 parts of ethylenediamine tetraacetate, 0.3-0.7 part of coconut diethanolamide and 120 parts of water 100-; the alkaline degreasing cleaning solution is prepared from the following raw materials in parts by weight: 10-15 parts of nicotine, 20-30 parts of dipropylene glycol, 3-5 parts of sodium oleate, 0.3-0.7 part of cocamidopropyl betaine and 120 parts of water.
Further, in the step S2, the mass ratio of the fish scales to the extracting solution is 1 (20-30); in the step S2, the mass ratio of the crude extract to the sodium chloride solution to the glacial acetic acid is 1 (20-30) to 10-15; the extracting solution is at least one of water, ethanol and ethyl acetate; the decolorizing agent is selected from at least one of activated carbon, fly ash and chitosan.
The preparation method combines the advantages of various fish collagen preparation methods in the prior art, the fish collagen has higher yield, better purity and lower preparation cost, the raw materials used in the process are wide in source, the preparation process is simple and easy to operate, the preparation method is suitable for large-scale production, and the prepared collagen product has high quality and high yield; before extraction, degreasing cleaning solutions with different properties are adopted for cleaning, the cleaning effect is better, lipid dissolution is facilitated, and the later extraction is promoted, so that the prepared fish collagen has higher purity, the preparation process is safe, sanitary and harmless, and the social value and the economic value are higher.
The invention will be further described with reference to specific examples, but the scope of protection of the invention is not limited thereto:
example 1
The present example provides a method for preparing fish collagen, comprising the steps of:
step S1, fish scale pretreatment: adding fish phosphorus into water containing white vinegar and salt, soaking and softening for 2 hours at 35 ℃, then adding an acidic degreasing cleaning solution for rinsing for 20 minutes, then adding an alkaline degreasing cleaning solution for rinsing for 15 minutes, then rinsing with distilled water to neutrality, finely cutting fish scales, washing with ethanol for 3 times, putting into a stirrer for stirring, washing with 0.03mol/L Tris-HCl buffer solution containing 4mol/L NaCl for 1 hour, and finally centrifuging to take precipitates; the mass ratio of the fish phosphorus to the white vinegar to the salt to the water to the acidic degreasing cleaning solution to the alkaline degreasing cleaning solution is 1:0.2:0.1:10:8: 10;
step S2, extraction: adding the fish scales prepared in the step S1 into water, carrying out shearing extraction at 45 ℃ to ensure that the grain diameter of the fish scales reaches 50 micrometers, then carrying out rotary evaporation to remove an extracting solution to obtain a crude extract, adding a sodium chloride solution with the mass fraction of 0.3% and glacial acetic acid with the mass fraction of 0.5mol/L into the crude extract, stirring for 1 hour, then adding a decoloring agent to carry out decoloring and deodorization, and taking supernatant to obtain an extraction stock solution; the mass ratio of the fish scales to the extracting solution is 1: 20; the mass ratio of the crude extract to the sodium chloride solution to the glacial acetic acid is 1:20: 10; the decolorizing agent is chitosan;
step S3, purification and post-treatment: slowly adding 4mol/L sodium carbonate into the extraction stock solution prepared in the step S2 to adjust the pH value to 7, centrifuging to remove precipitates, then filling the rest solution into a dialysis bag, respectively dialyzing for 1d by using acetic acid solutions with the mass fractions of 0.1% and 0.01%, dialyzing for 2d by using distilled water, changing the dialyzate twice every day, and finally obtaining the fish collagen aqueous solution with higher purity; the purified collagen aqueous solution is prepared into a pure fish collagen sample by adopting a low-temperature freeze drying method.
The acidic degreasing cleaning solution is prepared from the following raw materials in parts by weight: 5 parts of glycyrrhizic acid, 10 parts of citric acid, 3 parts of ethylenediamine tetraacetate, 0.3 part of coconut diethanolamide and 100 parts of water.
The alkaline degreasing cleaning solution is prepared from the following raw materials in parts by weight: 10 parts of nicotine, 20 parts of dipropylene glycol, 3 parts of sodium oleate, 0.3 part of cocamidopropyl betaine and 100 parts of water.
Example 2
This example provides a method for producing fish collagen, which is substantially the same as in example 1, except that in step S1, the mass ratio of fish phosphorus, white vinegar, salt, water, an acidic degreasing cleaning solution and an alkaline degreasing cleaning solution is 1:0.25:0.15:13:8.5: 10.5; the acidic degreasing cleaning solution is prepared from the following raw materials in parts by weight: 6.5 parts of glycyrrhizic acid, 11.5 parts of citric acid, 3.5 parts of ethylenediamine tetraacetate, 0.4 part of coconut diethanolamide and 105 parts of water; the alkaline degreasing cleaning solution is prepared from the following raw materials in parts by weight: 11 parts of nicotine, 22 parts of dipropylene glycol, 3.5 parts of sodium oleate, 0.4 part of cocamidopropyl betaine and 105 parts of water.
In the step S2, the mass ratio of the fish scales to the extracting solution is 1: 23; the mass ratio of the crude extract, the sodium chloride solution and the glacial acetic acid in the step S2 is 1:23: 11; the extracting solution is ethanol; the decolorizing agent is fly ash.
Example 3
This example provides a method for producing fish collagen, which is substantially the same as in example 1, except that, in step S1, the mass ratio of fish phosphorus, white vinegar, salt, water, acidic degreasing cleaning solution and alkaline degreasing cleaning solution is 1:0.3:0.2:15:9: 11; the acidic degreasing cleaning solution is prepared from the following raw materials in parts by weight: 7 parts of glycyrrhizic acid, 13 parts of citric acid, 4 parts of ethylene diamine tetraacetic acid, 0.5 part of coconut diethanolamide and 110 parts of water; the alkaline degreasing cleaning solution is prepared from the following raw materials in parts by weight: 13 parts of nicotine, 25 parts of dipropylene glycol, 4 parts of sodium oleate, 0.5 part of cocamidopropyl betaine and 110 parts of water.
In the step S2, the mass ratio of the fish scales to the extracting solution is 1: 25; the mass ratio of the crude extract, the sodium chloride solution and the glacial acetic acid in the step S2 is 1:25: 13; the extracting solution is ethyl acetate; the decolorizing agent is activated carbon.
Example 4
This example provides a method for producing fish collagen, which is substantially the same as in example 1, except that, in step S1, the mass ratio of fish phosphorus, white vinegar, salt, water, an acidic degreasing cleaning solution to an alkaline degreasing cleaning solution is 1:0.35:0.25:18:9.5: 11.5; the acidic degreasing cleaning solution is prepared from the following raw materials in parts by weight: 9 parts of glycyrrhizic acid, 14 parts of citric acid, 4.5 parts of ethylenediamine tetraacetate, 0.6 part of coconut diethanolamide and 118 parts of water; the alkaline degreasing cleaning solution is prepared from the following raw materials in parts by weight: 14.5 parts of nicotine, 29 parts of dipropylene glycol, 4.5 parts of sodium oleate, 0.6 part of cocamidopropyl betaine and 118 parts of water.
In the step S2, the mass ratio of the fish scales to the extracting solution is 1: 28; the mass ratio of the crude extract, the sodium chloride solution and the glacial acetic acid in the step S2 is 1:28: 14.5; the extracting solution is formed by mixing water, ethanol and ethyl acetate according to the mass ratio of 1:2: 3; the decolorizing agent is formed by mixing activated carbon, fly ash and chitosan according to the mass ratio of 2:1: 3.
Example 5
This example provides a process for the preparation of fish collagen, which is essentially identical to that of example 1, except that: further, in the step S1, the mass ratio of the fish phosphorus, the white vinegar, the salt, the water, the acidic degreasing cleaning solution and the alkaline degreasing cleaning solution is 1:0.4:0.3:20:8: 10; the acidic degreasing cleaning solution is prepared from the following raw materials in parts by weight: 10 parts of glycyrrhizic acid, 15 parts of citric acid, 5 parts of ethylene diamine tetraacetic acid, 0.7 part of coconut diethanolamide and 120 parts of water; the alkaline degreasing cleaning solution is prepared from the following raw materials in parts by weight: 15 parts of nicotine, 30 parts of dipropylene glycol, 5 parts of sodium oleate, 0.7 part of cocamidopropyl betaine and 120 parts of water.
In the step S2, the mass ratio of the fish scales to the extracting solution is 1: 30; the mass ratio of the crude extract, the sodium chloride solution and the glacial acetic acid in the step S2 is 1:30: 15; the extracting solution is ethanol; the decolorizing agent is chitosan.
Comparative example 1
This example provides a process for the preparation of fish collagen, which is essentially identical to that of example 1, except that: no acidic degreasing cleaning solution was used.
Comparative example 2
This example provides a process for the preparation of fish collagen, which is essentially identical to that of example 1, except that: no alkaline degreasing cleaning solution was used.
Comparative example 3
This example provides a process for the preparation of fish collagen which is essentially the same as that of example 1 except that no decolorizing agent is used for decolorization.
Comparative example 4
This example provides a process for the preparation of fish collagen, which is essentially identical to that of example 1, except that: no dialysis treatment was used.
To further illustrate the technical effects of the examples of the present invention, the fish collagen prepared by the examples 1 to 5 of the present invention and the comparative examples 1 to 4 was tested, and the test results are shown in table 1.
TABLE 1
| Detecting items | Percentage of produced flour (%) | Purity (%) | Digestibility (%) |
| Example 1 | 63 | 98.5 | 95 |
| Example 2 | 65 | 98.7 | 96 |
| Example 3 | 66 | 99.0 | 98 |
| Example 4 | 68 | 99.3 | 97 |
| Example 5 | 70 | 99.7 | 99 |
| Comparative example 1 | 50 | 92.0 | 92 |
| Comparative example 2 | 51 | 90.5 | 91 |
| Comparative example 3 | 52 | 91.1 | 90 |
| Comparative example 4 | 50 | 90.0 | 90 |
As can be seen from the above table, the fish collagen prepared by the method for preparing fish collagen disclosed in the embodiments of the present invention has higher yield and purity, and is easier to digest.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (8)
1. The efficient preparation method of the fish collagen is characterized by comprising the following steps:
step S1, fish scale pretreatment: adding fish phosphorus into water containing acetic acid and salt, soaking and softening for 2-3 hours at 35-45 ℃, then adding an acidic degreasing cleaning solution for rinsing for 20-30 minutes, then adding an alkaline degreasing cleaning solution for rinsing for 15-25 minutes, then rinsing with distilled water to neutrality, then finely cutting fish scales, washing with ethanol for 3-5 times, putting into a stirrer for stirring, washing with 0.03-0.06mol/L Tris-HCl buffer solution containing 4-6mol/L NaCl for 1-2 hours, and finally centrifuging to obtain a precipitate;
step S2, extraction: adding the fish scales prepared in the step S1 into the extracting solution, carrying out shearing extraction at 30-60 ℃ to enable the grain size of the fish scales to reach 10-100 mu m, then carrying out rotary evaporation to remove the extracting solution to obtain a crude extract, adding a sodium chloride solution with the mass fraction of 0.3-0.5% and 0.5mol/L of glacial acetic acid into the crude extract, stirring for 1-2 hours, then adding a decolorizing agent to carry out decolorization and deodorization, and taking supernatant to obtain an extraction stock solution;
step S3, purification and post-treatment: slowly adding 4-6mol/L sodium carbonate into the extraction stock solution prepared in the step S2 to adjust the pH value to 7, centrifuging to remove precipitates, then filling the rest solution into a dialysis bag, respectively dialyzing for 1-2d by using acetic acid solutions with the mass fractions of 0.1% and 0.01%, dialyzing for 2-3d by using distilled water, changing the dialyzate twice every day, and finally obtaining the fish collagen aqueous solution with higher purity; the purified collagen aqueous solution is prepared into a pure fish collagen sample by adopting a low-temperature freeze drying method.
2. The method for efficiently preparing fish collagen according to claim 1, wherein the mass ratio of the fish phosphorus, the white vinegar, the salt, the water, the acidic degreasing cleaning solution and the alkaline degreasing cleaning solution in step S1 is 1 (0.2-0.4): 0.1-0.3): 10-20): 8-10): 10-12.
3. The efficient preparation method of fish collagen according to claim 1, wherein the acidic degreasing cleaning solution is prepared from the following raw materials in parts by weight: 5-10 parts of glycyrrhizic acid, 10-15 parts of citric acid, 3-5 parts of ethylenediamine tetraacetate, 0.3-0.7 part of coconut diethanolamide and 120 parts of water 100-.
4. The efficient preparation method of fish collagen according to claim 1, wherein the alkaline degreasing cleaning solution is prepared from the following raw materials in parts by weight: 10-15 parts of nicotine, 20-30 parts of dipropylene glycol, 3-5 parts of sodium oleate, 0.3-0.7 part of cocamidopropyl betaine and 120 parts of water.
5. The method for efficiently preparing fish collagen according to claim 1, wherein the mass ratio of the fish scales to the extracting solution in step S2 is 1 (20-30).
6. The method for efficiently preparing fish collagen according to claim 1, wherein the mass ratio of the crude extract, the sodium chloride solution and the glacial acetic acid in step S2 is 1 (20-30) to (10-15).
7. The method for efficiently producing fish collagen according to claim 1, wherein the extract is at least one of water, ethanol, and ethyl acetate.
8. The method for efficiently preparing fish collagen according to claim 1, wherein the decolorant is at least one selected from the group consisting of activated carbon, fly ash and chitosan.
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