CN117987497A - Preparation method of fish-source collagen peptide - Google Patents
Preparation method of fish-source collagen peptide Download PDFInfo
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- CN117987497A CN117987497A CN202311857505.5A CN202311857505A CN117987497A CN 117987497 A CN117987497 A CN 117987497A CN 202311857505 A CN202311857505 A CN 202311857505A CN 117987497 A CN117987497 A CN 117987497A
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- 108010035532 Collagen Proteins 0.000 title claims abstract description 129
- 229920001436 collagen Polymers 0.000 title claims abstract description 129
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 53
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 241000251468 Actinopterygii Species 0.000 claims abstract description 120
- 239000002131 composite material Substances 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 37
- 239000002253 acid Substances 0.000 claims abstract description 27
- -1 alcohol compound Chemical class 0.000 claims abstract description 23
- 150000001875 compounds Chemical class 0.000 claims abstract description 23
- 238000000605 extraction Methods 0.000 claims abstract description 22
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 20
- 102000057297 Pepsin A Human genes 0.000 claims abstract description 15
- 108090000284 Pepsin A Proteins 0.000 claims abstract description 15
- 239000003513 alkali Substances 0.000 claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- 229940111202 pepsin Drugs 0.000 claims abstract description 15
- 239000000284 extract Substances 0.000 claims abstract description 10
- 238000001914 filtration Methods 0.000 claims abstract description 9
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 235000019688 fish Nutrition 0.000 claims description 118
- 239000000243 solution Substances 0.000 claims description 78
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- 238000004140 cleaning Methods 0.000 claims description 24
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 15
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 239000012535 impurity Substances 0.000 claims description 10
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 235000002639 sodium chloride Nutrition 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- UWJJYHHHVWZFEP-UHFFFAOYSA-N pentane-1,1-diol Chemical compound CCCCC(O)O UWJJYHHHVWZFEP-UHFFFAOYSA-N 0.000 claims description 8
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- 238000004519 manufacturing process Methods 0.000 claims description 7
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- 238000000265 homogenisation Methods 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 5
- 235000013372 meat Nutrition 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
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- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
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- XTEGARKTQYYJKE-UHFFFAOYSA-N chloric acid Chemical compound OCl(=O)=O XTEGARKTQYYJKE-UHFFFAOYSA-N 0.000 claims description 4
- 229940005991 chloric acid Drugs 0.000 claims description 4
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 4
- 239000012267 brine Substances 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 2
- 241000972773 Aulopiformes Species 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 244000247747 Coptis groenlandica Species 0.000 claims description 2
- 235000002991 Coptis groenlandica Nutrition 0.000 claims description 2
- 241000252230 Ctenopharyngodon idella Species 0.000 claims description 2
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 2
- 241000630524 Taractes rubescens Species 0.000 claims description 2
- 241000276707 Tilapia Species 0.000 claims description 2
- 241001125843 Trichiuridae Species 0.000 claims description 2
- 235000019270 ammonium chloride Nutrition 0.000 claims description 2
- 239000000908 ammonium hydroxide Substances 0.000 claims description 2
- RQPZNWPYLFFXCP-UHFFFAOYSA-L barium dihydroxide Chemical compound [OH-].[OH-].[Ba+2] RQPZNWPYLFFXCP-UHFFFAOYSA-L 0.000 claims description 2
- 229910001863 barium hydroxide Inorganic materials 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 235000011148 calcium chloride Nutrition 0.000 claims description 2
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 claims description 2
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- 229910001861 calcium hydroxide Inorganic materials 0.000 claims description 2
- 241001233037 catfish Species 0.000 claims description 2
- 239000013505 freshwater Substances 0.000 claims description 2
- 238000004255 ion exchange chromatography Methods 0.000 claims description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 2
- 239000001103 potassium chloride Substances 0.000 claims description 2
- 235000011164 potassium chloride Nutrition 0.000 claims description 2
- 239000008213 purified water Substances 0.000 claims description 2
- 235000019515 salmon Nutrition 0.000 claims description 2
- 239000013535 sea water Substances 0.000 claims description 2
- 239000011592 zinc chloride Substances 0.000 claims description 2
- 235000005074 zinc chloride Nutrition 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 15
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 4
- 230000004071 biological effect Effects 0.000 abstract description 4
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- 102000012422 Collagen Type I Human genes 0.000 description 3
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- 102000016942 Elastin Human genes 0.000 description 1
- 108010014258 Elastin Proteins 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
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 206010072170 Skin wound Diseases 0.000 description 1
- 210000001361 achilles tendon Anatomy 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
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- 210000000845 cartilage Anatomy 0.000 description 1
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- 210000004207 dermis Anatomy 0.000 description 1
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- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of biological materials, in particular to a preparation method of a fish collagen peptide. The preparation method comprises the following steps: adding fish body tissue into the first composite solution, extracting collagen at 2-15 ℃, and filtering to obtain a crude collagen extract; wherein the first composite solution comprises a first acid compound, a first alcohol compound and pepsin; further purifying the obtained crude collagen extract to obtain refined collagen; adding the obtained refined collagen into a second composite solution to react at the temperature of 2-15 ℃, and homogenizing under high pressure in the reaction process; the second composite solution comprises a second acid compound and a second glycol compound. The low-temperature acid alcohol extraction method used in the preparation method provided by the invention avoids the problem of biological activity reduction caused by the change of collagen structure by a hydrolysis method, an enzyme method and an alkali method, effectively ensures the extraction rate of protein and reduces the microbial content of the product.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a preparation method of a fish collagen peptide.
Background
Collagen is a major component of animal connective tissue, a fibrous structure protein, and is widely present in animal skin, cartilage, achilles tendon, and other tissues. Collagen is a triple helix structure consisting of three alpha peptide chains, with a molecular weight of about 300kDa. Collagen contains a large number of amino acids, of which hydroxyproline is a characteristic amino acid. Collagen is of a wide variety, and is commonly available in types I, II, III, V and XI. Type i collagen is the most abundant and important class of collagen in animals. The fish skin, fish scales, fish bones and other fish body tissues also contain rich type I collagen, and the collagen extracted from fish scraps realizes the reutilization of wastes, so that the environmental pollution can be reduced. Compared with collagen extracted from mammals, the fish-derived collagen reduces the risk of zoonosis, is easier to absorb, and has better biocompatibility, degradability and bioactivity.
Collagen has certain application in cosmetics industry, medicine industry and food industry. Collagen stimulates dermal fibroblasts to generate new collagen and tissues, has the effects of repairing and regenerating, can be used as a raw material to be added into cosmetics, and can also be prepared into dressing to promote repairing skin wounds. Collagen plays a positive role in guiding the regeneration process of bone tissue, can form a biological barrier membrane at the bone defect part, and creates good conditions for the proliferation of regenerated tissue cells. In addition, the collagen can be used as a functional substance and a nutritional ingredient to be added into food, so that good elasticity of skin is enhanced and maintained.
To ensure good efficacy of collagen in cosmetics, it must be made to function in the dermis layer of the skin. The factors influencing the functional expression of the collagen are mainly the molecular weight, and macromolecular collagen is not easily absorbed by skin. The methods for reducing the molecular weight of collagen mainly comprise hydrolysis, acid degradation, alkali degradation and enzyme degradation. Patent publication No. CN104278069A discloses a method for obtaining protein by enzymolysis at 37 ℃, which has higher extraction temperature and is easy to reduce the activity of the protein. The publication No. CN109480294A discloses a method for obtaining protein by high-temperature hydrolysis, the temperature is 130-135 ℃, and the protein is easy to inactivate. Patent publication No. CN104351849A discloses a method for extracting protein by alkali dissolution, which is easy to denature collagen and generate racemization phenomenon, thereby reducing the activity of protein.
Disclosure of Invention
The invention provides a preparation method of fish collagen peptide, which avoids the problem of biological activity reduction caused by changing the collagen structure by hydrolysis method, enzyme method and alkaline method by using a low-temperature acid alcohol extraction method, effectively ensures the extraction rate of protein and reduces the microbial content of the product.
According to a first aspect of the present invention, there is provided a method for preparing a collagen peptide of fish origin, comprising the steps of:
Step (1): adding fish body tissue into the first composite solution, extracting collagen at 2-15 ℃, and filtering to obtain a crude collagen extract; wherein the first composite solution comprises a first acid compound, a first alcohol compound and pepsin;
Step (2): further purifying the crude collagen extract obtained in the step (1) to obtain refined collagen;
Step (3): adding the obtained refined collagen into a second composite solution to react at the temperature of 2-15 ℃, and homogenizing under high pressure in the reaction process; wherein the second composite solution comprises a second acid compound and a second glycol compound.
In the scheme, the preparation method of the fish collagen peptide comprises the steps of carrying out collagen enzymolysis and preliminary extraction on fish tissues by adopting a first composite solution containing a first acid compound, a first alcohol compound and pepsin, wherein the first acid compound is beneficial to the dissolution of collagen from the fish tissues, the pepsin can remove the terminal peptide of the collagen, the dissolution rate of the collagen is improved, the bioactivity of the collagen can be ensured by adding the first alcohol compound, and the microbial content can be effectively reduced. According to the preparation method disclosed by the invention, after the collagen is extracted, the crude collagen extracting solution is further purified, so that the purity of the collagen can be improved, and the refined collagen with high purity can be obtained. According to the preparation method, after purification, the obtained refined collagen is added into a second composite solution comprising a second acid compound and a second glycol compound to react at the temperature of 2-15 ℃, so that collagen peptide in the refined collagen is further effectively extracted, the extraction efficiency is improved, and meanwhile, high-pressure homogenization is carried out in the process of the refined collagen reaction, so that the fish-source collagen peptide with stable molecular weight distribution range and concentrated molecular weight is obtained. The preparation method of the invention is simple, has low cost, is suitable for wide production and application, and can obtain the fish collagen peptide with high bioactivity, stable molecular weight distribution range, high yield and low cost.
In order to achieve higher extraction efficiency, further, in the step (1), the first acid compound includes one or more of citric acid, acetic acid, hydrochloric acid, carbonic acid, phosphoric acid and chloric acid; and/or the first alcohol compound comprises one or more of pentanediol, butanediol, glycerol and ethylene glycol;
Preferably, in the first composite solution, the concentration of the first acid compound is 0.1-1.0 mol/L; and/or, in the first composite solution, the mass percentage of the first alcohol compound is 1% -5%; and/or, in the first composite solution, the mass percentage of the pepsin is 0.01% -0.1%. The concentration of the first acid compound, the mass percentage of the first alcohol compound and the mass percentage of the pepsin are limited within reasonable range values, so that the acid, the alcohol and the protease in the first compound solution achieve better synergistic interaction, and the extraction efficiency is better improved.
In order to achieve higher extraction efficiency, in step (1), collagen extraction is performed for 12-48 hours.
In order to achieve higher extraction efficiency, further, in the step (3), the second acid compound includes one or more of citric acid, acetic acid, hydrochloric acid, carbonic acid, phosphoric acid and chloric acid; preferably, in the second composite solution, the concentration of the second acid compound is 0.001-0.01 mol/L;
And/or the second glycol compound comprises one or more of pentanediol, butanediol, glycerol and ethylene glycol; preferably, in the second composite solution, the mass percentage of the second glycol compound is 1% -5%.
Further, in the step (3), the mass-volume ratio of the refined collagen to the second composite solution is 1:100-1:500.
In the above-described aspect, the extraction efficiency of collagen can be further improved by limiting the mass-to-volume ratio of the purified collagen to the second composite solution to a reasonable range value.
Further, the reaction time is 24-96 hours.
In the above scheme, the activity of the collagen peptide can be further ensured by limiting the reaction time to a reasonable range value so that the refined collagen fully reacts with the second composite solution.
Further, the high pressure homogenization pressure is 5000-15000 psi.
In the scheme, the pressure of high-pressure homogenization is limited within a reasonable range, so that the fish-source collagen peptide with stable molecular weight distribution range and concentrated molecular weight is more beneficial to be obtained.
Further, in step (2), the purification method comprises one or more of salting-out and reconstitution, ion chromatography exchange, ultrafiltration, filtration or drying.
In the above scheme, the purity of the fish collagen peptide can be improved by selecting a proper purification method.
Further, step (3) further comprises: and after the reaction is finished, regulating the pH value of the solution to 5.5-8.5.
In the scheme, the pH value of the solution is regulated to a reasonable range value after the reaction is finished, so that the subsequent treatment and application of the fish-source collagen peptide are facilitated.
Further, the step (1) further comprises the step of pre-treating the fish body tissue before adding the fish body tissue into the first composite solution: firstly, cleaning redundant fish meat, fish fat and macroscopic impurities on the surface of the fish body tissue, and then putting the fish body tissue into a cleaning solution for cleaning and removing impurities and removing water.
In the scheme, before the fish body tissue is added into the first composite solution, the pretreatment step is carried out on the fish body tissue, so that impurities in the fish body tissue are removed, and the quality of the fish-derived collagen peptide is improved.
Preferably, the cleaning time is 24-96 hours; and/or the weight ratio of the fish body tissue to the cleaning solution is 1:30-1:100.
In the scheme, the cleaning time and/or the weight ratio of the fish body tissue to the cleaning solution are limited within a reasonable range, so that the efficiency of removing impurities in the fish body tissue can be improved, and the quality of the fish-derived collagen peptide can be further improved.
Further, the cleaning solution comprises one or more of a saline solution, an alkaline solution or single purified water;
Preferably, the salt in the salt water solution comprises one or more of potassium chloride, sodium chloride, calcium chloride, zinc chloride, ferric chloride and ammonium chloride; and/or the alkali in the alkali aqueous solution comprises one or more of sodium hydroxide, potassium hydroxide, ammonium hydroxide, barium hydroxide and calcium hydroxide;
more preferably, the mass concentration of salt in the brine solution is 0.5% -10%; and/or, the mass concentration of the alkali in the alkali aqueous solution is 0.1% -1%.
In the scheme, the aim of removing impurities in the fish body tissues more effectively can be achieved by selecting a cleaning solution with reasonable types and mass concentration.
Further, in the step (1), the fish body tissue includes a fish skin, a fish scale or a fish bone; the fish in the fish body tissue is one of a sea water fish and a freshwater fish; preferably, the fish in the fish body tissue is selected from one or more of cod, pomfret, weever, tilapia, salmon, gold thread fish, crucian, grass carp, catfish, silver carp, bighead carp or hairtail.
The raw materials adopted by the preparation method are fish body tissues such as the fish skin, the fish scales, the fish bones and the like, so that the treatment of fish offal is solved, the reutilization of wastes is realized, and the environmental pollution is reduced.
The invention has the beneficial effects that:
The preparation method of the fish-source collagen peptide can prepare the high-purity fish-source collagen peptide with high bioactivity and stable molecular weight distribution range by adopting a low-temperature acid alcohol extraction method, can avoid the problem of reduced bioactivity caused by changing the collagen structure by a hydrolysis method, an enzyme method or an alkali method, can effectively ensure the extraction rate of protein, and reduces the microbial content of the product.
The raw materials adopted by the preparation method of the fish-source collagen peptide are fish body tissues such as fish skin, fish scales and fish bones, so that the treatment of fish offal is solved, the reutilization of wastes is realized, and the environmental pollution is reduced.
Compared with the collagen peptide extracted from mammals, the fish-derived collagen peptide prepared by the preparation method reduces the risk of zoonosis, is easier to absorb, and has very good biocompatibility and degradability.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides a preparation method of a fish collagen peptide, which comprises the following steps:
step (1): the fish meat, fish fat and macroscopic impurities on the surface of the fish body tissue are cleaned by a scraper. Crushing the fish body tissue into small blocks by using a slicer, putting the small blocks into 10% potassium chloride salt solution for cleaning, wherein the ratio of the fish body tissue to the cleaning solution is 1:100, the cleaning time is 24 hours, and then removing water by using a centrifuge.
The cleaned fish body tissue is put into a compound solution containing 1.0mol/L acetic acid of 0.1 percent pepsin and 1 percent pentanediol to react for 36 hours at the temperature of 2-15 ℃, and the crude extract of the collagen is obtained by filtering.
Step (2): and (3) further purifying the crude collagen extract by an ultrafiltration system and a drying system in sequence to obtain refined collagen.
Step (3): the purified refined collagen is mixed with 0.01mol/L acetic acid and 1% pentanediol compound solution according to the proportion of 1:100, and reacts for 48 hours at the temperature of 2-15 ℃, and a high-pressure homogenizer acts on the collagen liquid with the pressure of 15000psi in the reaction process. And after the reaction is finished, regulating the pH value of the collagen liquid to 5.5-8.5.
Example 2
The embodiment provides a preparation method of a fish collagen peptide, which comprises the following steps:
Step (1): the fish meat, fish fat and macroscopic impurities on the surface of the fish body tissue are cleaned by a scraper. Crushing the fish body tissue into small blocks by using a slicing machine, sequentially adding the small blocks into a 0.5% sodium chloride solution and a 0.1% sodium hydroxide alkali solution for cleaning, wherein the ratio of the fish body tissue to the cleaning solution is 1:30, the cleaning time is 96 hours, and removing water by using a centrifuge.
The cleaned fish body tissue is put into acetic acid solution containing 0.03 percent pepsin and 0.5mol/L and pentanediol compound solution with 5 percent pepsin to react for 48 hours at the temperature of 2-15 ℃, and the crude extract of the collagen is obtained by filtering.
Step (2): salting out, re-dissolving, drying and further purifying the crude collagen extract to obtain refined collagen.
Step (3): the purified refined collagen is mixed with 0.001mol/L hydrochloric acid and 1% pentanediol compound solution according to the proportion of 1:500, and reacts for 96 hours at the temperature of 2-15 ℃, and a high-pressure homogenizer acts on the collagen liquid under the pressure of 5000psi in the reaction process. And adjusting the pH value of the collagen liquid to 5.5-8.5 by using acetic acid solution after shearing.
Example 3
The embodiment provides a preparation method of a fish collagen peptide, which comprises the following steps:
Step (1): the fish meat, fish fat and macroscopic impurities on the surface of the fish body tissue are cleaned by a scraper. Crushing the fish body tissue into small blocks by using a slicer, cleaning the small blocks by using a 1% potassium hydroxide alkali solution, wherein the ratio of the fish body tissue to the cleaning solution is 1:30, the cleaning time is 24 hours, and then removing water by using a centrifuge.
The cleaned fish body tissue is put into a compound solution containing 0.01 percent pepsin and 0.1mol/L citric acid and 5 percent butanediol to react for 12 hours at the temperature of 2-15 ℃, and the crude extract of the collagen is obtained by filtering.
Step (2): salting out, re-dissolving, drying and further purifying the crude collagen extract to obtain refined collagen.
Step (3): mixing the purified refined collagen solution with a 0.005mol/L citric acid solution and a 5% butanediol compound solution according to a ratio of 1:300, reacting for 24 hours at a temperature of between 2 and 15 ℃, and reacting the collagen solution with a high-pressure homogenizer at a pressure of 10000psi in the reaction process. And after the reaction is finished, regulating the pH of the collagen liquid to 6.5-8.5.
Example 4
The present example provides a method for preparing a fish-derived collagen peptide, which is different from example 1 in that the same amount of glycerol is used instead of pentanediol in the composite solution in step (1) and the composite solution in step (3).
Example 5
The present embodiment provides a method for preparing a fish-derived collagen peptide, which is different from embodiment 1 in that butanediol is used instead of pentanediol in the same amount in the composite solution in step (1) and the composite solution in step (3).
Example 6
The present example provides a method for preparing a collagen peptide of fish origin, which is different from example 3 in that in step (2), filtration is used instead of salting-out and reconstitution, and the rest steps are the same as those in example 3.
Example 7
The present example provides a method for preparing a fish collagen peptide, which is different from example 3 in that in step (2), molecular sieve chromatography and ion exchange column separation are used instead of salting-out and reconstitution, and the rest steps are the same as example 3.
The following performance tests were performed on the fish-derived collagen peptides obtained in examples 1 to 7:
distribution test of protein molecular weight: the detection is carried out by adopting the general rule 0541 electrophoresis method and the MALDI-TOF-MS method of the pharmacopoeia of the people's republic of China (2020 edition).
And (3) testing the extraction rate of the fish-source collagen peptide: the protein content is measured by adopting a method for measuring the protein content in general rule 0731 of pharmacopoeia of the people's republic of China (2020 edition).
Purity test of fish collagen peptide: the purity of the collagen is determined by adopting a YY/T1453-2016 type I collagen characterization method of a tissue engineering medical device product.
Biological activity of fish collagen peptide and microbiological condition test of product: the biological activity of the product is evaluated by taking keratinocytes and fibroblasts as a test system and detecting the changes of cell mobility, active oxygen relative inhibition rate and fibroblast elastin content. The microbiological condition of the product is evaluated according to the microbiological test method of the fifth chapter of cosmetic safety specifications (2015 edition).
The test results obtained are shown in table 1 below.
TABLE 1
As can be seen from the results in Table 1, the preparation method of the invention can obtain the fish-derived collagen peptide with molecular weight distribution of 0.5-10kDa, extraction rate of more than 8%, purity of up to 99% and obvious bioactivity, and can effectively reduce colony growth quantity of the fish-derived collagen peptide.
Comparative example 1
The comparative example provides a method for producing a collagen peptide of fish origin, which is different from example 1 in that a high-pressure homogenizer is not used to act on the collagen liquid in step (3), and the rest of the steps are the same as in example 1.
Comparative example 2
This comparative example provides a method for producing a collagen peptide of fish origin, which is different from example 1 in that 0.01mol/L acetic acid is not added during the reaction in step (3), and the rest of the steps are the same as in example 1.
The fish-derived collagen peptides obtained in comparative examples 1 and 2 were subjected to a distribution test of protein molecular weight, and the test results are shown in table 2 below.
TABLE 2
| Project | Molecular weight distribution |
| Example 1 | The molecular weight of the protein is concentrated between 0.5 and 2kDa |
| Comparative example 1 | The molecular weight of the protein is concentrated between 75 and 100kDa |
| Comparative example 2 | The molecular weight of the protein is concentrated between 100 kDa and 240kDa |
As can be seen from the results of Table 2, the addition of the acid solution and the use of the high-pressure homogenization step in the further reaction process of the refined collagen in the step (3) of the preparation method of the fish-derived collagen peptide have great influence on the molecular weight distribution of the final fish-derived collagen peptide, and the addition of the acid solution and the high-pressure homogenization step are favorable for obtaining the stable fish-derived collagen peptide with the molecular weight concentrated at 0.5-2 kDa.
Comparative example 3
This comparative example provides a method for preparing a collagen peptide of fish origin, which is different from example 2 in that the complex solution in step (1) does not contain an acetic acid solution, and the rest of the steps are the same as example 2.
Comparative example 4
This comparative example provides a method for preparing a collagen peptide of fish origin, which is different from example 2 in that the complex solution in step (1) does not contain pepsin, and the rest of the steps are the same as example 2.
The fish-derived collagen peptides obtained in comparative example 3 and comparative example 4 were subjected to an extraction rate test, and the results obtained are shown in table 3 below.
TABLE 3 Table 3
| Project | Collagen extraction yield (%) |
| Example 2 | 10.2 |
| Comparative example 3 | 3.3 |
| Comparative example 4 | 5.6 |
As can be seen from the results in Table 3, the addition of the acid compound and pepsin in the preparation process of the crude collagen extract in the step (1) of the preparation method of the fish-derived collagen peptide can influence the extraction rate of the final fish-derived collagen peptide, and the fish-derived collagen peptide with high extraction rate can be obtained in the environment of 2-15 ℃ through the mutual matching of the acid compound, the alcohol compound and the pepsin.
Comparative example 5
The comparative example provides a method for preparing a collagen peptide of fish origin, which is different from example 1 in that the reaction temperature in both step (1) and step (3) is 37 ℃.
Comparative example 6
This comparative example provides a method for producing a collagen peptide of fish origin, which is different from example 1 in that the composite solution in step (1) and the composite solution in step (3) do not contain pentanediol.
The bioactivity of the fish-derived collagen peptides of comparative example 5 and comparative example 6 and the microbial status of the products were tested, and the test results are shown in table 4 below.
TABLE 4 Table 4
As can be seen from the results of table 4, the fish-derived collagen peptide prepared by the preparation method of example 1 exhibited significant bioactive advantages over comparative examples 5 and 6, and effectively reduced the colony growth number.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The preparation method of the fish collagen peptide is characterized by comprising the following steps:
Step (1): adding fish body tissue into the first composite solution, extracting collagen at 2-15 ℃, and filtering to obtain a crude collagen extract; wherein the first composite solution comprises a first acid compound, a first alcohol compound and pepsin;
Step (2): further purifying the crude collagen extract obtained in the step (1) to obtain refined collagen;
Step (3): adding the obtained refined collagen into a second composite solution to react at the temperature of 2-15 ℃, and homogenizing under high pressure in the reaction process; wherein the second composite solution comprises a second acid compound and a second glycol compound.
2. The production method according to claim 1, wherein in the step (1), the first acid-based compound includes one or more of citric acid, acetic acid, hydrochloric acid, carbonic acid, phosphoric acid, and chloric acid; and/or the first alcohol compound comprises one or more of pentanediol, butanediol, glycerol and ethylene glycol;
Preferably, in the first composite solution, the concentration of the first acid compound is 0.1-1.0 mol/L; and/or, in the first composite solution, the mass percentage of the first alcohol compound is 1% -5%; and/or, in the first composite solution, the mass percentage of the pepsin is 0.01% -0.1%.
3. The method according to claim 1 or 2, wherein in the step (1), the collagen extraction is performed for 12 to 48 hours.
4. The production method according to claim 1, wherein in the step (3), the second acid-based compound includes one or more of citric acid, acetic acid, hydrochloric acid, carbonic acid, phosphoric acid, and chloric acid; preferably, in the second composite solution, the concentration of the second acid compound is 0.001-0.01 mol/L;
And/or the second glycol compound comprises one or more of pentanediol, butanediol, glycerol and ethylene glycol; preferably, in the second composite solution, the mass percentage of the second glycol compound is 1% -5%.
5. The method according to claim 1 or 4, wherein in the step (3), the mass-to-volume ratio of the purified collagen to the second composite solution is 1:100 to 1:500;
And/or the reaction time is 24-96 h;
And/or the high pressure homogenization pressure is 5000-15000 psi.
6. The method of claim 1, wherein in step (2), the purification method comprises one or more of salting-out reconstitution, ion-chromatography exchange, ultrafiltration, filtration, or drying; preferably, the purification method comprises salting out reconstitution and drying.
7. The method of manufacturing according to claim 1, wherein step (3) further comprises: and after the reaction is finished, regulating the pH value of the solution to 5.5-8.5.
8. The method of claim 1, wherein step (1) further comprises pre-treating the fish body tissue prior to adding the fish body tissue to the first composite solution: firstly, cleaning redundant fish meat, fish fat and macroscopic impurities on the surface of a fish body tissue, and then putting the fish body tissue into a cleaning solution for cleaning and removing impurities and removing water;
Preferably, the cleaning time is 24-96 hours; and/or the weight ratio of the fish body tissue to the cleaning solution is 1:30-1:100.
9. The method of claim 8, wherein the cleaning solution comprises one or more of a brine solution, an aqueous alkali solution, or single purified water;
Preferably, the salt in the salt water solution comprises one or more of potassium chloride, sodium chloride, calcium chloride, zinc chloride, ferric chloride and ammonium chloride; and/or the alkali in the alkali aqueous solution comprises one or more of sodium hydroxide, potassium hydroxide, ammonium hydroxide, barium hydroxide and calcium hydroxide;
more preferably, the mass concentration of salt in the brine solution is 0.5% -10%; and/or, the mass concentration of the alkali in the alkali aqueous solution is 0.1% -1%.
10. The method of claim 1, wherein in step (1), the fish body tissue comprises fish skin, fish scale, or fish bone; the fish in the fish body tissue is one of a sea water fish and a freshwater fish; preferably, the fish in the fish body tissue is selected from one or more of cod, pomfret, weever, tilapia, salmon, gold thread fish, crucian, grass carp, catfish, silver carp, bighead carp or hairtail.
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