CN103388017A - Preparation technology of fish skin collagen oligopeptide - Google Patents
Preparation technology of fish skin collagen oligopeptide Download PDFInfo
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Abstract
The invention discloses a preparation technology of fish skin collagen oligopeptide. The preparation technology comprises the steps of: firstly, pre-treating fish skin with acid and alkali to remove fat and other impurities, thus obtaining a collagen protein extract with high purity, wherein the extraction rate of collagen protein is higher than 85%; then, performing enzymolysis on the collagen protein extract by using a two-step enzymolysis method; at the end of enzymolysis, purifying enzymatic hydrolysate by a centrifugal machine cooperating with a multistage membrane separating system to obtain a fish skin collagen oligopeptide solution; finally, rapidly drying the fish skin collagen oligopeptide solution in a spray manner to obtain collagen oligopeptide powder with purity being higher than 90% and molecular weight being smaller than 1000 Da. The collagen oligopeptide powder prepared by the technology is low in cost, high in yield and purity, and suitable to large-scaled production.
Description
Technical field
The present invention relates to a kind of preparation technology of fish skin collagen oligopeptides, belong to biological technical field.
Background technology
Fish-skin is the by product of fish processing, and its protein content is very high, particularly the content of collagen protein account for its total protein half also want many.Utilize the small molecules collagen oligopeptide that the protease hydrolysis collagen protein obtains to have unique physiologically active, as: promote skin collagen metabolism, preventing osteoporosis, antitumor, improve immunity, suppress ACE etc., can be widely used in makeup, medicine, a plurality of fields such as functional food.Simultaneously,, due to the epiphytotics generations such as mad cow disease, foot and mouth disease, bird flu in recent years, consider security and the Cost Problems of raw material, adopt fish-skin to prepare collagen oligopeptide and have more application prospect and value.
In recent years, along with the using value of collagen oligopeptide more and more is subject to people's attention, its correlative study also more a report.The report for preparing collagen oligopeptide with animal skin, as the patent of invention of CN1434124A, disclosing with pigskin collagen is raw material, utilize Sumizyme MP and aspartic protease or and the synchronious hydrolysis of neutral protease obtain the method for oligopeptides.The preparation of fish skin collagen oligopeptides such as the application for a patent for invention of CN103060414A, a kind of method that biological enzyme prepares the fish skin collagen oligopeptides is disclosed, its step comprises pre-treatment, enzymic hydrolysis, takes off peculiar smell, extractant regeneration, decolouring concentrate, join the thing drying and other steps, the collagen oligopeptide protein content that makes is 50%～53%, moisture 5%～5.5%, molecular-weight average are 250～750 Da.Han Fengjie etc. (food science and technology, 2006,5:130-132) provide a kind of method with the standby oligopeptides of flatfish leather, the flatfish skin is through pre-treatment, and enzymolysis has obtained the little peptide of molecular weight distribution at 0.6～1.8 kDa.The above-mentioned method safety for preparing collagen oligopeptide is easy, but the oligopeptides sample protein content of preparation is low, and molecular weight distribution is wider, is difficult for absorbing.For obtaining high collagen content, the concentrated collagen oligopeptide of molecular weight, the present invention has set up a kind of new fish skin collagen oligopeptides production technique.
Summary of the invention
Technical problem to be solved by this invention is to provide the preparation technology of a kind of simple and safe, easy handling, fish skin collagen oligopeptides that cost product purity low, that produce is high, to take full advantage of the by product of fish processing, turn waste into wealth, the fish skin collagen oligopeptides that exploitation has broad prospect of application.
For solving the problems of the technologies described above, technical solution of the present invention is:
A kind of preparation technology of fish skin collagen oligopeptides, is characterized in that comprising the steps:
(1) pre-treatment:
After fish-skin is cleaned with clear water, drop in reactor, add the alkaline solution of 5～8 times of fish-skin quality, the concentration of alkaline solution is 0.001～0.01Kg/L, stirs 1～3 hour, is washed to neutrality; The acid solution that adds again 5～8 times of fish-skin quality, the volumetric concentration of acid solution are 0.1～1%, stir 1～3 hour, are washed to neutrality; The water that adds finally 1～2 times of fish-skin quality,, in 60 ℃, heated 2～4 hours, obtains the collagen of fish skin extracting solution;
(2) two step enzymolysis:
In above-mentioned fish-skin extracting solution, add the proteolytic enzyme that is equivalent to fish-skin quality 1～30% to carry out the first step enzymolysis, hydrolysis temperature is 30～60 ℃, enzyme digestion reaction pH value is controlled at 6～8, enzymolysis 3～12 hours; Be warming up to 80～85 ℃ after enzymolysis finishes, constant temperature 10～20min enzyme that goes out, obtain primary enzymolysis liquid; Add the aspartic protease that is equivalent to fish-skin quality 1～30% to carry out the second step enzymolysis in primary enzymolysis liquid, hydrolysis temperature is 40～60 ℃ again, and enzyme digestion reaction pH value is controlled at 3～6, enzymolysis 3～12 hours; Be warming up to 80～85 ℃ after enzymolysis finishes, constant temperature 10～20min enzyme that goes out, obtain secondary enzymolysis liquid;
(3) purifying is concentrated:
Remove the solid residue of secondary enzymolysis liquid with whizzer; The gained clear liquid is removed a small amount of macromole impurity with the ultra-filtration membrane that molecular weight is 1000 Da～8000Da, then with the nanofiltration membrane that molecular weight is 200 Da, removes inorganic salt and small molecular weight impurity and concentrated, obtains the fish skin collagen oligopeptide solution;
(4) rapid drying:
Spraying drying fish skin collagen oligopeptide solution, obtain fish skin collagen oligopeptides powder.
The alkali that described step (1) pre-treatment is used is sodium bicarbonate or sodium hydroxide, and acid used is hydrochloric acid.
In described step (2), the first step enzymolysis proteolytic enzyme used is a kind of or any two kinds of combinations of trypsinase, flavor protease, fish skin and scale Collagen Hydrolysate enzyme (being called for short fish skin and scale proteolytic enzyme), sea-food proteolytic enzyme.
Regulating pH alkali used in described step (2) is sodium bicarbonate or sodium hydroxide, and acid used is hydrochloric acid.
In described step (3), whizzer is the whizzer of 10000～15000 rpm.
The ultra-filtration membrane that uses in described step (3), film pressure are 0.5～1MPa, and working temperature is 25～40 ℃; The nanofiltration membrane of using, film pressure are 1～2 MPa, and working temperature is 25～40 ℃.
While using nanofiltration membrane in described step (3), concentrated secondary enzymolysis liquid until original volume 5%～20%.
In described step (4), spray-dired air outlet temperature is 70～90 ℃, and inlet temperature is 140～180 ℃.
In described step (4), the purity of fish skin collagen oligopeptides powder is greater than 90%, and molecular weight is less than 1000 Da, and color is white or faint yellow, free from extraneous odour.
After adopting such scheme, the present invention has following beneficial effect: this process makes full use fish processing byproduct fish-skin prepares collagen oligopeptide, and with low cost, collagen yield is greater than 85%; Adopt centrifugal collaborative Using Multistage Membranes separation system purifying fish skin collagen oligopeptides, removal of impurities and concentrated effect are good, are fit to large-scale industrial production; The fish skin collagen oligopeptides of preparation is white or pale yellow powder, and purity is greater than 90%, and molecular weight is less than 1000 Da, free from extraneous odour.
Embodiment
The invention will be further described below in conjunction with specific embodiment.
Embodiment one:
1. pre-treatment:, take the high-quality fish-skin as raw material, can be ocean class fish-skin, can be also the freshwater fish fish-skin.After 5 kg fish-skins are cleaned with clear water, drop in reactor, adding 30 L concentration is the aqueous sodium hydroxide solution of 0.002 Kg/L, stirs after 2 hours, is washed to neutrality; Adding 30 L volumetric concentrations is 0.6% aqueous hydrochloric acid again, stirs after 1 hour, is washed to neutrality; The water that adds finally 5 L,, in 60 ℃, heated 4 hours, obtains the collagen of fish skin extracting solution;
2. enzymolysis: in above-mentioned collagen of fish skin extracting solution, add the trypsinase of 0.05 kg to carry out the first step enzymolysis, hydrolysis temperature is 40 ℃, regulate pH 7.0 with sodium hydroxide solution, enzymolysis 6 hours, enzymolysis is warming up to 80 ℃ after finishing, the constant temperature 20 min enzyme that goes out, obtain primary enzymolysis liquid; Add the aspartic protease of 0.05 kg to carry out the second step enzymolysis in primary enzymolysis liquid, hydrolysis temperature is 50 ℃, with hydrochloric acid soln, regulates pH3.5 again, enzymolysis 6 hours; Be warming up to 80 ℃ after enzymolysis finishes, the constant temperature 20 min enzyme that goes out, obtain secondary enzymolysis liquid;
3. purifying is concentrated: the solid residue of removing secondary enzymolysis liquid with the whizzer of 10000 rpm.The gained clear liquid is removed a small amount of macromole impurity with the ultra-filtration membrane that molecular weight is 2000 Da, and film pressure is 0.7 MPa, and working temperature is 35 ℃; Remove inorganic salt and small molecular weight impurity with the nanofiltration membrane that molecular weight is 200 Da again, film pressure is 1.5 MPa, and working temperature is 35 ℃, concentrated secondary enzymolysis liquid to 3 L, obtaining purity is 95%, and molecular weight is less than 1000 Da, and molecular-weight average is the fish skin collagen oligopeptide solution of 600 Da;
4. rapid drying: spraying drying fish skin collagen oligopeptide solution, air outlet temperature are 80 ℃, and inlet temperature is 160 ℃, obtain fish skin collagen oligopeptides powder.
Embodiment two:
1. pre-treatment:, take the high-quality fish-skin as raw material, can be ocean class fish-skin, can be also the freshwater fish fish-skin.After 5 kg fish-skins are cleaned with clear water, drop in reactor, adding 30 L concentration is the sodium bicarbonate aqueous solution of 0.01 Kg/L, stirs after 2 hours, is washed to neutrality; Adding 30 L volumetric concentrations is 0.8% aqueous hydrochloric acid again, stirs after 1 hour, is washed to neutrality; The water that adds finally 5 L,, in 60 ℃, heated 4 hours, obtains the collagen of fish skin extracting solution;
2. enzymolysis: in above-mentioned collagen of fish skin extracting solution, add the fish skin and scale Collagen Hydrolysate enzyme of 0.1 kg to carry out the first step enzymolysis, hydrolysis temperature is 50 ℃, regulate pH 6.5 with sodium hydroxide solution, enzymolysis 4 hours, be warming up to 85 ℃ after enzymolysis finishes, the constant temperature 15 min enzyme that goes out, obtain primary enzymolysis liquid; Add the aspartic protease of 0.2 kg to carry out the second step enzymolysis in primary enzymolysis liquid, hydrolysis temperature is 50 ℃, with hydrochloric acid soln, regulates pH3.0 again, enzymolysis 4 hours; Be warming up to 85 ℃ after enzymolysis finishes, the constant temperature 15 min enzyme that goes out, obtain secondary enzymolysis liquid;
3. purifying is concentrated: the solid residue of removing secondary enzymolysis liquid with the whizzer of 13000 rpm.The gained clear liquid is removed a small amount of macromole impurity with the ultra-filtration membrane that molecular weight is 3500 Da, and film pressure is 0.8 MPa, and working temperature is 30 ℃; Remove inorganic salt and small molecular weight impurity with the nanofiltration membrane that molecular weight is 200 Da again, film pressure is 1.5 MPa, and working temperature is 30 ℃, concentrated secondary enzymolysis liquid to 3 L, obtaining purity is 90%, and molecular weight is less than 1000 Da, and molecular-weight average is the fish skin collagen oligopeptide solution of 800 Da;
4. rapid drying: spraying drying fish skin collagen oligopeptide solution, air outlet temperature are 70 ℃, and inlet temperature is 160 ℃, obtain fish skin collagen oligopeptides powder.
Embodiment three:
1. pre-treatment:, take the high-quality fish-skin as raw material, can be ocean class fish-skin, can be also the freshwater fish fish-skin.After 5 kg fish-skins are cleaned with clear water, drop in reactor, adding 40 L concentration is the aqueous sodium hydroxide solution of 0.004 Kg/L, stirs after 2 hours, is washed to neutrality; Adding 40 L volumetric concentrations is 1% aqueous hydrochloric acid again, stirs after 1 hour, is washed to neutrality; The water that adds finally 5 L,, in 60 ℃, heated 2 hours, obtains the collagen of fish skin extracting solution;
2. enzymolysis: in above-mentioned collagen of fish skin extracting solution, add the trypsinase of 0.5 kg and the sea-food proteolytic enzyme of 0.5 kg to carry out the first step enzymolysis, hydrolysis temperature is 50 ℃, regulate pH 7.5 with sodium hydroxide solution, enzymolysis 10 hours, be warming up to 80 ℃ after enzymolysis finishes, the constant temperature 20 min enzyme that goes out, obtain primary enzymolysis liquid; Add the aspartic protease of 1 kg to carry out the second step enzymolysis in primary enzymolysis liquid, hydrolysis temperature is 50 ℃, with hydrochloric acid soln, regulates pH5.0 again, enzymolysis 10 hours; Be warming up to 80 ℃ after enzymolysis finishes, the constant temperature 20 min enzyme that goes out, obtain secondary enzymolysis liquid;
3. purifying is concentrated: the solid residue of removing secondary enzymolysis liquid with the whizzer of 15000 rpm.The gained clear liquid is removed a small amount of macromole impurity with the ultra-filtration membrane that molecular weight is 8000 Da, and film pressure is 0.6 MPa, and working temperature is 30 ℃; Remove inorganic salt and small molecular weight impurity with the nanofiltration membrane that molecular weight is 200 Da again, film pressure is 1.5 MPa, and working temperature is 30 ℃, concentrated secondary enzymolysis liquid to 3 L, obtaining purity is 92%, and molecular weight is less than 1000 Da, and molecular-weight average is the fish skin collagen oligopeptide solution of 400 Da;
4. rapid drying: spraying drying fish skin collagen oligopeptide solution, air outlet temperature are 90 ℃, and inlet temperature is 180 ℃, obtain fish skin collagen oligopeptides powder.
Embodiment four:
1. pre-treatment:, take the high-quality fish-skin as raw material, can be ocean class fish-skin, can be also the freshwater fish fish-skin.After 10 kg fish-skins are cleaned with clear water, drop in reactor, adding 50 L concentration is the sodium bicarbonate aqueous solution of 0.008 Kg/L, stirs after 3 hours, is washed to neutrality; Adding 50 L volumetric concentrations is 1% aqueous hydrochloric acid again, stirs after 3 hours, is washed to neutrality; The water that adds finally 10 L,, in 60 ℃, heated 2 hours, obtains the collagen of fish skin extracting solution;
2. enzymolysis: in above-mentioned collagen of fish skin extracting solution, add the trypsinase of 0.2 kg and the flavor protease of 0.2 kg to carry out the first step enzymolysis, hydrolysis temperature is 50 ℃, regulate pH 7.0 with sodium hydroxide solution, enzymolysis 4 hours, be warming up to 80 ℃ after enzymolysis finishes, the constant temperature 20 min enzyme that goes out, obtain primary enzymolysis liquid; Add the aspartic protease of 0.5 kg to carry out the second step enzymolysis in primary enzymolysis liquid, hydrolysis temperature is 50 ℃, with hydrochloric acid soln, regulates pH3.0 again, enzymolysis 4 hours; Be warming up to 80 ℃ after enzymolysis finishes, the constant temperature 20 min enzyme that goes out, obtain secondary enzymolysis liquid;
3. purifying is concentrated: the solid residue of removing secondary enzymolysis liquid with the whizzer of 13000 rpm.The gained clear liquid is removed a small amount of macromole impurity with the ultra-filtration membrane that molecular weight is 5000 Da, and film pressure is 0.9 MPa, and working temperature is 35 ℃; Remove inorganic salt and small molecular weight impurity with the nanofiltration membrane that molecular weight is 200 Da again, film pressure is 1.5 MPa, and working temperature is 30 ℃, concentrated secondary enzymolysis liquid to 5 L, obtaining purity is 91%, and molecular weight is less than 1000 Da, and molecular-weight average is the fish skin collagen oligopeptide solution of 500 Da;
4. rapid drying: spraying drying fish skin collagen oligopeptide solution, air outlet temperature are 80 ℃, and inlet temperature is 180 ℃, obtain fish skin collagen oligopeptides powder.
Embodiment five:
1. pre-treatment:, take the high-quality fish-skin as raw material, can be ocean class fish-skin, can be also the freshwater fish fish-skin.After 10 kg fish-skins are cleaned with clear water, drop in reactor, adding 80 L concentration is the aqueous sodium hydroxide solution of 0.003 Kg/L, stirs after 2 hours, is washed to neutrality; Adding 80 L volumetric concentrations is 1% aqueous hydrochloric acid again, stirs after 1 hour, is washed to neutrality; The water that adds finally 10 L,, in 60 ℃, heated 2 hours, obtains the collagen of fish skin extracting solution;
2. enzymolysis: in above-mentioned collagen of fish skin extracting solution, add the trypsinase of 2 kg to carry out the first step enzymolysis, hydrolysis temperature is 50 ℃, regulate pH 7.0 with sodium hydroxide solution, enzymolysis 8 hours, enzymolysis is warming up to 80 ℃ after finishing, the constant temperature 20 min enzyme that goes out, obtain primary enzymolysis liquid; Add the aspartic protease of 1 kg to carry out the second step enzymolysis in primary enzymolysis liquid, hydrolysis temperature is 50 ℃ again, with hydrochloric acid soln, regulates pH 6, enzymolysis 8 hours; Be warming up to 80 ℃ after enzymolysis finishes, the constant temperature 20 min enzyme that goes out, obtain secondary enzymolysis liquid;
3. purifying is concentrated: the solid residue of removing secondary enzymolysis liquid with the whizzer of 10000 rpm.The gained clear liquid is removed a small amount of macromole impurity with the ultra-filtration membrane that molecular weight is 1000 Da, and film pressure is 0.6 MPa, and working temperature is 35 ℃; Remove inorganic salt and small molecular weight impurity with the nanofiltration membrane that molecular weight is 200 Da again, film pressure is 1.5 MPa, and working temperature is 35 ℃, concentrated secondary enzymolysis liquid to 6 L, obtaining purity is 92%, and molecular weight is less than 1000 Da, and molecular-weight average is the fish skin collagen oligopeptide solution of 600 Da;
4. rapid drying: spraying drying fish skin collagen oligopeptide solution, air outlet temperature are 80 ℃, and inlet temperature is 160 ℃, obtain fish skin collagen oligopeptides powder.
The above, be only preferred embodiment of the present invention, not technical scope of the present invention is imposed any restrictions, thus variation or the modification in every case according to claim of the present invention and specification sheets, done, within all should belonging to the scope that patent of the present invention contains.
Claims (9)
1. the preparation technology of a fish skin collagen oligopeptides, is characterized in that comprising the steps:
(1) pre-treatment:
, with soda acid pre-treatment fish-skin,, except degrease and other impurity, obtain the collagen protein extracting solution;
(2) two step enzymolysis:
In above-mentioned collagen of fish skin extracting solution, add the proteolytic enzyme that is equivalent to fish-skin quality 1～30% to carry out the first step enzymolysis, hydrolysis temperature is 30～60 ℃, enzyme digestion reaction pH value is controlled at 6～8, enzymolysis 3～12 hours; Be warming up to 80～85 ℃ after enzymolysis finishes, constant temperature 10～20min enzyme that goes out, obtain primary enzymolysis liquid; Add the aspartic protease that is equivalent to fish-skin quality 1～30% to carry out the second step enzymolysis in primary enzymolysis liquid, hydrolysis temperature is 40～60 ℃ again, and enzyme digestion reaction pH value is controlled at 3～6, enzymolysis 3～12 hours; Be warming up to 80～85 ℃ after enzymolysis finishes, constant temperature 10～20min enzyme that goes out, obtain secondary enzymolysis liquid;
(3) purifying is concentrated:
Remove the solid residue of secondary enzymolysis liquid with whizzer; The gained clear liquid is removed a small amount of macromole impurity with the ultra-filtration membrane that molecular weight is 1000 Da～8000Da, then with the nanofiltration membrane that molecular weight is 200 Da, removes inorganic salt and small molecular weight impurity and concentrated, obtains the fish skin collagen oligopeptide solution;
(4) rapid drying:
Spraying drying fish skin collagen oligopeptide solution, obtain fish skin collagen oligopeptides powder.
2. the preparation technology of fish skin collagen oligopeptides according to claim 1, it is characterized in that: described step (1) pre-treatment detailed process is: fish-skin is dropped in reactor, the alkaline solution that adds 5～8 times of fish-skin quality, the concentration of alkaline solution is 0.001～0.01 Kg/L, stirred 1～3 hour, and be washed to neutrality; The acid solution that adds again 5～8 times of fish-skin quality, the volumetric concentration of acid solution are 0.1～1%, stir 1～3 hour, are washed to neutrality; The water that adds finally 1～2 times of fish-skin quality,, in 60 ℃, heated 2～4 hours, obtains the collagen of fish skin extracting solution.
3. the preparation technology of fish skin collagen oligopeptides according to claim 1, it is characterized in that: the alkali that described step (1) pre-treatment is used is sodium bicarbonate or sodium hydroxide, and acid used is hydrochloric acid.
4. the preparation technology of fish skin collagen oligopeptides according to claim 1, it is characterized in that: in described step (2), the first step enzymolysis proteolytic enzyme used is a kind of or any two kinds of combinations of trypsinase, flavor protease, fish skin and scale Collagen Hydrolysate enzyme, sea-food proteolytic enzyme.
5. the preparation technology of fish skin collagen oligopeptides according to claim 1, it is characterized in that: regulating pH alkali used in described step (2) is sodium bicarbonate or sodium hydroxide, and acid used is hydrochloric acid.
6. the preparation technology of fish skin collagen oligopeptides according to claim 1, it is characterized in that: in described step (3), whizzer is the whizzer of 10000～15000 rpm.
7. the preparation technology of fish skin collagen oligopeptides according to claim 1, it is characterized in that: the ultra-filtration membrane that uses in described step (3), film pressure are 0.5～1 MPa, working temperature is 25～40 ℃; The nanofiltration membrane of using, film pressure are 1～2 MPa, and working temperature is 25～40 ℃.
8. the preparation technology of fish skin collagen oligopeptides according to claim 1 is characterized in that: while using nanofiltration membrane in described step (3), concentrated secondary enzymolysis liquid until original volume 5%～20%.
9. the preparation technology of fish skin collagen oligopeptides according to claim 1, it is characterized in that: in described step (4), spray-dired air outlet temperature is 70～90 ℃, inlet temperature is 140～180 ℃.
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