CN103388017B - 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 preparation technology who the present invention relates to a kind of fish skin collagen oligopeptides, belongs 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 protease hydrolysis collagen protein obtains to there is 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.Meanwhile, due to epiphytotics generations such as mad cow disease, foot and mouth disease, bird flus 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 is more and more subject to people's attention, its correlative study also more a report.The report of preparing collagen oligopeptide by 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 is as the application for a patent for invention of CN103060414A, disclose a kind of biological enzyme and prepared the method for fish skin collagen oligopeptides, its step comprises that pre-treatment, enzymic hydrolysis, de-peculiar smell, extractant regeneration, decolouring concentrate, join thing drying and other steps, the collagen oligopeptide protein content making is 50%～53%, moisture 5%～5.5%, molecular-weight average is 250～750 Da.Han Fengjie etc. (food science and technology, 2006,5:130-132) provide a kind of flatfish leather of using for the method for oligopeptides, 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 of 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 make full use of the by product of fish processing, turn waste into wealth, exploitation has the fish skin collagen oligopeptides of broad prospect of application.
For solving the problems of the technologies described above, technical solution of the present invention is:
A preparation technology for 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 is 0.1～1%, stirs 1～3 hour, is washed to neutrality; The water that finally adds 1～2 times of fish-skin quality, in 60 ℃, heats 2～4 hours, obtains 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; After enzymolysis finishes, be warming up to 80～85 ℃, constant temperature 10～20min enzyme that goes out, obtains primary enzymolysis liquid; In primary enzymolysis liquid, add the aspartic protease that is equivalent to fish-skin quality 1～30% to carry out second step enzymolysis, hydrolysis temperature is 40～60 ℃ again, and enzyme digestion reaction pH value is controlled at 3～6, enzymolysis 3～12 hours; After enzymolysis finishes, be warming up to 80～85 ℃, constant temperature 10～20min enzyme that goes out, obtains secondary enzymolysis liquid;
(3) purifying is concentrated:
With whizzer, remove the solid residue of secondary enzymolysis liquid; Gained clear liquid is removed a small amount of macromole impurity with the ultra-filtration membrane that molecular weight is 1000 Da～8000Da, then removes inorganic salt and small molecular weight impurity concentrated by the nanofiltration membrane that molecular weight is 200 Da, obtains fish skin collagen oligopeptide solution;
(4) rapid drying:
Spray dried fish collagen oligopeptide solution, obtains 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.
In described step (2), regulating pH alkali used 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 using in described step (3), film pressure is 0.5～1MPa, working temperature is 25～40 ℃; The nanofiltration membrane of using, film pressure is 1～2 MPa, 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.
Adopt after such scheme, the present invention has following beneficial effect: this process makes full use fish processing byproduct fish-skin is prepared 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 applicable to large-scale industrial and produce; 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
Below in conjunction with specific embodiment, the invention will be further described.
embodiment mono-:
1. pre-treatment: take high-quality fish-skin as raw material, can be ocean class fish-skin, can be also 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; Add again the aqueous hydrochloric acid that 30 L volumetric concentrations are 0.6%, stir after 1 hour, be washed to neutrality; The water that finally adds 5 L, in 60 ℃, heats 4 hours, obtains 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 ℃, with sodium hydroxide solution, regulate pH 7.0, enzymolysis 6 hours, is warming up to 80 ℃ after enzymolysis finishes, the constant temperature 20 min enzyme that goes out, obtains primary enzymolysis liquid; In primary enzymolysis liquid, add the aspartic protease of 0.05 kg to carry out second step enzymolysis, hydrolysis temperature is 50 ℃, with hydrochloric acid soln, regulates pH3.5 again, enzymolysis 6 hours; After enzymolysis finishes, be warming up to 80 ℃, the constant temperature 20 min enzyme that goes out, obtains secondary enzymolysis liquid;
3. purifying is concentrated: the solid residue of removing secondary enzymolysis liquid with the whizzer of 10000 rpm.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 ℃; By the nanofiltration membrane that molecular weight is 200 Da, remove inorganic salt and small molecular weight impurity 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: spray dried fish collagen oligopeptide solution, air outlet temperature is 80 ℃, inlet temperature is 160 ℃, obtains fish skin collagen oligopeptides powder.
embodiment bis-:
1. pre-treatment: take high-quality fish-skin as raw material, can be ocean class fish-skin, can be also 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; Add again the aqueous hydrochloric acid that 30 L volumetric concentrations are 0.8%, stir after 1 hour, be washed to neutrality; The water that finally adds 5 L, in 60 ℃, heats 4 hours, obtains 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 ℃, with sodium hydroxide solution, regulate pH 6.5, enzymolysis 4 hours, after enzymolysis finishes, be warming up to 85 ℃, the constant temperature 15 min enzyme that goes out, obtains primary enzymolysis liquid; In primary enzymolysis liquid, add the aspartic protease of 0.2 kg to carry out second step enzymolysis, hydrolysis temperature is 50 ℃, with hydrochloric acid soln, regulates pH3.0 again, enzymolysis 4 hours; After enzymolysis finishes, be warming up to 85 ℃, the constant temperature 15 min enzyme that goes out, obtains secondary enzymolysis liquid;
3. purifying is concentrated: the solid residue of removing secondary enzymolysis liquid with the whizzer of 13000 rpm.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 ℃; By the nanofiltration membrane that molecular weight is 200 Da, remove inorganic salt and small molecular weight impurity 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: spray dried fish collagen oligopeptide solution, air outlet temperature is 70 ℃, inlet temperature is 160 ℃, obtains fish skin collagen oligopeptides powder.
embodiment tri-:
1. pre-treatment: take high-quality fish-skin as raw material, can be ocean class fish-skin, can be also 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; Add again the aqueous hydrochloric acid that 40 L volumetric concentrations are 1%, stir after 1 hour, be washed to neutrality; The water that finally adds 5 L, in 60 ℃, heats 2 hours, obtains 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 ℃, with sodium hydroxide solution, regulate pH 7.5, enzymolysis 10 hours, after enzymolysis finishes, be warming up to 80 ℃, the constant temperature 20 min enzyme that goes out, obtains primary enzymolysis liquid; In primary enzymolysis liquid, add the aspartic protease of 1 kg to carry out second step enzymolysis, hydrolysis temperature is 50 ℃, with hydrochloric acid soln, regulates pH5.0 again, enzymolysis 10 hours; After enzymolysis finishes, be warming up to 80 ℃, the constant temperature 20 min enzyme that goes out, obtains secondary enzymolysis liquid;
3. purifying is concentrated: the solid residue of removing secondary enzymolysis liquid with the whizzer of 15000 rpm.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 ℃; By the nanofiltration membrane that molecular weight is 200 Da, remove inorganic salt and small molecular weight impurity 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: spray dried fish collagen oligopeptide solution, air outlet temperature is 90 ℃, inlet temperature is 180 ℃, obtains fish skin collagen oligopeptides powder.
embodiment tetra-:
1. pre-treatment: take high-quality fish-skin as raw material, can be ocean class fish-skin, can be also 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; Add again the aqueous hydrochloric acid that 50 L volumetric concentrations are 1%, stir after 3 hours, be washed to neutrality; The water that finally adds 10 L, in 60 ℃, heats 2 hours, obtains 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 ℃, with sodium hydroxide solution, regulate pH 7.0, enzymolysis 4 hours, after enzymolysis finishes, be warming up to 80 ℃, the constant temperature 20 min enzyme that goes out, obtains primary enzymolysis liquid; In primary enzymolysis liquid, add the aspartic protease of 0.5 kg to carry out second step enzymolysis, hydrolysis temperature is 50 ℃, with hydrochloric acid soln, regulates pH3.0 again, enzymolysis 4 hours; After enzymolysis finishes, be warming up to 80 ℃, the constant temperature 20 min enzyme that goes out, obtains secondary enzymolysis liquid;
3. purifying is concentrated: the solid residue of removing secondary enzymolysis liquid with the whizzer of 13000 rpm.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 ℃; By the nanofiltration membrane that molecular weight is 200 Da, remove inorganic salt and small molecular weight impurity 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: spray dried fish collagen oligopeptide solution, air outlet temperature is 80 ℃, inlet temperature is 180 ℃, obtains fish skin collagen oligopeptides powder.
embodiment five:
1. pre-treatment: take high-quality fish-skin as raw material, can be ocean class fish-skin, can be also 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; Add again the aqueous hydrochloric acid that 80 L volumetric concentrations are 1%, stir after 1 hour, be washed to neutrality; The water that finally adds 10 L, in 60 ℃, heats 2 hours, obtains 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 ℃, with sodium hydroxide solution, regulate pH 7.0, enzymolysis 8 hours, is warming up to 80 ℃ after enzymolysis finishes, the constant temperature 20 min enzyme that goes out, obtains primary enzymolysis liquid; In primary enzymolysis liquid, add the aspartic protease of 1 kg to carry out second step enzymolysis, hydrolysis temperature is 50 ℃ again, with hydrochloric acid soln, regulates pH 6, enzymolysis 8 hours; After enzymolysis finishes, be warming up to 80 ℃, the constant temperature 20 min enzyme that goes out, obtains secondary enzymolysis liquid;
3. purifying is concentrated: the solid residue of removing secondary enzymolysis liquid with the whizzer of 10000 rpm.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 ℃; By the nanofiltration membrane that molecular weight is 200 Da, remove inorganic salt and small molecular weight impurity 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: spray dried fish collagen oligopeptide solution, air outlet temperature is 80 ℃, inlet temperature is 160 ℃, obtains 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 (7)
1. a preparation technology for 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 collagen protein extracting solution, the alkali using is sodium bicarbonate or sodium hydroxide, acid used is hydrochloric acid;
(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; After enzymolysis finishes, be warming up to 80～85 ℃, constant temperature 10～20min enzyme that goes out, obtains primary enzymolysis liquid; In primary enzymolysis liquid, add the aspartic protease that is equivalent to fish-skin quality 1～30% to carry out second step enzymolysis, hydrolysis temperature is 40～60 ℃ again, and enzyme digestion reaction pH value is controlled at 3～6, enzymolysis 3～12 hours; After enzymolysis finishes, be warming up to 80～85 ℃, constant temperature 10～20min enzyme that goes out, obtains secondary enzymolysis liquid; 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;
(3) purifying is concentrated:
With whizzer, remove the solid residue of secondary enzymolysis liquid; Gained clear liquid is removed a small amount of macromole impurity with the ultra-filtration membrane that molecular weight is 1000 Da～8000Da, then removes inorganic salt and small molecular weight impurity concentrated by the nanofiltration membrane that molecular weight is 200 Da, obtains fish skin collagen oligopeptide solution;
(4) rapid drying:
Spray dried fish collagen oligopeptide solution, obtains 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, stir 1～3 hour, be washed to neutrality; The acid solution that adds again 5～8 times of fish-skin quality, the volumetric concentration of acid solution is 0.1～1%, stirs 1～3 hour, is washed to neutrality; The water that finally adds 1～2 times of fish-skin quality, in 60 ℃, heats 2～4 hours, obtains collagen of fish skin extracting solution.
3. the preparation technology of fish skin collagen oligopeptides according to claim 1, is characterized in that: in described step (2), regulating pH alkali 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, is characterized in that: in described step (3), whizzer is the whizzer of 10000～15000 rpm.
5. the preparation technology of fish skin collagen oligopeptides according to claim 1, is characterized in that: the ultra-filtration membrane using in described step (3), and film pressure is 0.5～1 MPa, working temperature is 25～40 ℃; The nanofiltration membrane of using, film pressure is 1～2 MPa, working temperature is 25～40 ℃.
6. 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%.
7. the preparation technology of fish skin collagen oligopeptides according to claim 1, is characterized in that: in described step (4), spray-dired air outlet temperature is 70～90 ℃, and inlet temperature is 140～180 ℃.
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