CN102251003B - Preparation technique of marine-organism-derived antihypertensive peptides - Google Patents
Preparation technique of marine-organism-derived antihypertensive peptides Download PDFInfo
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Abstract
The invention discloses a preparation technique of marine-organism-derived antihypertensive peptides, which comprises the following steps: (1) pretreating a marine organism byproduct to remove surface impurities and fat on the marine organism byproduct; (2) extracting marine-organism-derived proteins by an enzyme method; (3) separating and purifying the extracting solution by a membrane separation technique to obtain the marine-organism-derived proteins; (4) decomposing the marine-organism-derived proteins with protease, and deactivating the protease after the enzymolysis finishes, thereby obtaining a marine-organism-derived antihypertensive peptide solution; and (5) quickly drying the marine-organism-derived antihypertensive peptide solution by spray drying to obtain the finished marine-organism-derived antihypertensive peptide powder product of which the protein content is higher than 90%. By adopting the technique, the marine-organism-derived antihypertensive peptides have the advantages of low cost, high antihypertensive activity, low average relative molecular weight and high purity, fully utilizes the marine organism byproduct, changes wastes into valuables, and is suitable for large-scale industrial production.
Description
Technical field
The present invention relates to the preparation technology of marine organisms source step-down peptide, belong to biological technical field.
Background technology
Human beings'health in the hypertension serious threat, whole world hyperpietic has 9.72 hundred million people approximately, annual because of the dead people of hypertension up to 1,200 ten thousand, be used for the treatment of the expense of hypertension and complication thereof every year also up to last trillion dollars, the curative effect of the antihypertensive drugs of synthetic obtains the generally approval of medical circle, but produce multiple side effect in process of clinical application, high security step-down peptide becomes the focus of research.Booster system body Shen Su – hypertensin system (the renial angiotensin system of human body, be called for short RAS) and depressurizing system swash peptide and discharge enzyme – kinin system (kallikrein-kinin system, abbreviation KKS) and playing the part of important role aspect blood pressure regulation and the cardiovascular function.Zinc metallopeptidase Zace1 (ACE) reaches the effect of rising blood pressure by the regulation and control to RAS system and KKS system.The step-down peptide suppresses the ACE enzyme, and the angiotensin (Ang II) that makes angiotensin (Ang I) to be converted into to have strong boosting keeps having the biological activity of vasorelaxation action bradykinin simultaneously, thereby reaches the purpose of step-down.Because the singularity (as high pressure, low temperature etc.) of the marine eco-environment, make marine organisms source protein matter have composition and the sequence of multiple specific amino acid, expose the avtive spot of step-down after the hydrolysis of marine organisms source protein matter, thereby make marine organisms source activity peptide have antihypertensive effect.Simultaneously, marine organisms source protein bioactive peptide has the characteristics of low antigenicity, and is safe, has no side effect.Therefore marine organisms source protein matter is the important medicine source of preparation step-down peptide, and the exploitation of marine organisms source step-down peptide will become the important component part of hypertension non-drug therapy.
Step-down peptide in marine organisms source is studied the nineties in last century abroad widely, enters after 21 century, and domestic research about marine organisms source step-down peptide also more gets up.Be the patent of invention of CN102051399A as publication number, disclose the extracting method of blood pressure lowering peptide in a kind of tuna, be organized as raw material with tuna, adopt stomach en-or papain enzymolysis, after centrifugal, with macroporous resin or dextrane gel resin purification, obtain the step-down peptide product.Publication number is the patent of invention of CN101240312A, a kind of preparation method who comes from the fish-skin ace inhibitory peptide is disclosed, it is raw material with the tilapia fishskin, water is carried the fish-skin albumen in the tilapia fishskin, adopt papoid or neutral protease to be hydrolyzed, centrifuging, ion-exchange, concentrate, ultra-filtration membrane purifying, SephadexG-15 gel chromatography, lyophilize makes the ace inhibitory peptide product.These methods utilize proteolytic enzyme to prepare the step-down peptide, and the three wastes are few, are cleaning procedures, the Product Safety height of production.But the isolation technique that these methods relate to is mainly ion exchange resin chromatography, gel filtration chromatography method, and from the angle of product development, cost of development is too high, is unfavorable for suitability for industrialized production.
Summary of the invention
The objective of the invention is problems such as little at the production cost height, the throughput that exist among the present marine organisms source step-down peptide preparation technology, that energy consumption is big, the preparation technology of the marine organisms source step-down peptide that a kind of technology is simple and safe, cost is low, produce product purity height, molecular weight are low, activity is high is provided, to take full advantage of the marine organisms byproduct, turn waste into wealth the marine organisms source step-down peptide of exploitation high added value.
For solving the problems of the technologies described above, technical solution of the present invention is:
The preparation technology of marine organisms source step-down peptide comprises the steps:
(1) pre-treatment
The marine organisms byproduct is dropped in the cleaning machine, add the alkaline solution of 10 times of marine organisms byproduct weight, the quality volumetric concentration of alkaline solution is 1-5%, stirs 10-12 hour, washes; Add the acid solution that is equivalent to 10 times of marine organisms byproduct weight again, the concentration of volume percent of acid solution is 0.5-5%, stirs 8-10 hour, and washing is clean, removes fat and other impurity of marine organisms byproduct;
(2) extraction of marine organisms source protein matter
To place reactor through pre-treatment and the marine organisms byproduct of cleaning, add the water be equivalent to ten times of marine organisms byproduct weight and be equivalent to the feed intake proteolytic enzyme of weight 0.3-0.9% of marine organisms byproduct and carry out enzymolysis, hydrolysis temperature is 35-50 ℃, enzyme digestion reaction pH value is controlled at 1-9, enzymolysis 3-6 hour; Enzymolysis is warmed up to 80 ℃ after finishing, and is incubated 20 minutes enzymes that go out, and obtains marine organisms source protein matter;
(3) purifying of marine organisms source protein matter
Marine organisms source protein matter is placed ceramic membrane equipment, after filter bag is handled, carry out the micro-filtration removal of impurities, remove particulate contamination and the macromole impurity of sample; Get rid of small molecular weight impurity and inorganic salt with nanofiltration membrane again, obtain high-purity marine organisms source protein matter;
(4) preparation of marine organisms source step-down peptide
Add in high-purity marine organisms source protein matter and be equivalent to the feed intake proteolytic enzyme of weight 0.3-0.9% of albumen and carry out enzymolysis, hydrolysis temperature is 40-60 ℃, and enzyme digestion reaction pH value is controlled at 5-8, enzymolysis 3-6 hour; Enzymolysis is warmed up to 80 ℃ after finishing, and is incubated the 15-35 minute enzyme that goes out, and obtains marine organisms source step-down peptide solution;
(5) rapid drying
Utilize spraying drying rapid drying marine organisms source step-down peptide solution, obtain marine organisms source step-down peptide powder finished product.
The employed alkali of described step (1) pre-treatment is sodium bicarbonate or sodium hydroxide, and employed acid is sulfuric acid or hydrochloric acid.
Used proteolytic enzyme is a kind of in stomach en-, aspartic protease, the Sumizyme MP in the described step (2).
Regulating pH alkali in the described step (2) is sodium hydroxide; Regulating pH acid is sulfuric acid or hydrochloric acid.
The aperture of employed filter bag is 1-20 μ m in the described step (3).
The aperture of employed ceramic membrane is 0.05-0.1 μ m in the described step (3), and film pressure is 0.3MPa, and temperature is 30-35 ℃.
Employed nanofiltration membrane relative molecular mass is 300D-2kD in the described step (3), and film pressure is 0.9MPa, and temperature is 25-30 ℃.
Used proteolytic enzyme is a kind of or two kinds of combinations in papoid, bromeline, neutral protease, trypsinase, Chymotrypsin, fish scale fish-skin proteolytic enzyme, sea-food proteolytic enzyme, the flavor protease in the described step (4).
Regulating pH alkali in the described step (4) is sodium hydroxide; Regulating pH acid is sulfuric acid.
Spray-dired air outlet temperature is that 70-90 ℃, inlet temperature are 140-180 ℃ in the described step (5).
Described marine organisms byproduct is fish scale and fish-skin.
Before the described pre-treatment step, earlier the marine organisms byproduct is cleaned to remove granule foreign.
After adopting such scheme, compared with the prior art the present invention has the following advantages:
1, the present invention directly utilizes ceramic membrane purifying marine organisms source protein matter extracting solution; reach the effect of removing granule foreign and macromole impurity; change traditional whizzer and separate the collaborative integrated separation purifying technique technology of membrane sepn; simplify technology; the shortening time; save cost, the separation efficiency height is beneficial to mass-producing and amplifies production.
2, the present invention utilizes nanofiltration membrane separation purifying marine organisms source protein matter; concentrate without vacuum concentrator; reach the effect of removing small molecular weight impurity and concentrating sample; good separating effect; obtain protein content and surpass 90% marine organisms source protein matter; simplify technology simultaneously, reduce energy consumption, save cost, be beneficial to large-scale industrial production.
3, the present invention utilizes enzyme engineering technology to prepare the step-down peptide, and the condition temperature is quality controllable, obtains the averagemolecular wt amount and surpasses 90% marine organisms source step-down peptide less than 5KD, protein content.
In a word, after the present invention adopted above-mentioned technology, obtained marine organisms source step-down peptide cost was low, antihypertensive activity is high, the averagemolecular wt amount is low, purity is high, and has taken full advantage of the marine organisms byproduct, turned waste into wealth, and was fit to large-scale industrial production.
Below in conjunction with the drawings and specific embodiments the present invention is further described.
Description of drawings
Fig. 1 is process flow sheet of the present invention.
Embodiment
Disclosed is the preparation technology of a kind of marine organisms source step-down peptide, as shown in Figure 1,---washing---is removed fat and other impurity---Enzymatic Extraction marine organisms source protein matter---membrane separation technique separation and purification marine organisms source protein matter---protease hydrolyzed marine organisms source protein matter---rapid drying marine organisms source step-down peptide of marine organisms byproduct to the present invention includes following steps: the marine organisms byproduct.
Below by the specific embodiment explanation.
Example one:
1, with the high-quality fish scale as raw material, selected fish scale can be that the marine fishes fish scale also can be the freshwater fish fish scale.Get fish scale 500kg and drop in the stirring and washing machine, add the water of 5000kg, clean 3 times, remove granule foreign.Fish scale after the washing is dropped in the cleaning machine, and adding 5000kg quality volumetric concentration is 5% sodium hydrogen carbonate solution, stirs 11 hours, washes.Adding the 5000kg concentration of volume percent is 5% hydrochloric acid soln, stirs 8 hours, washes, and removes viscous substance, fat and other impurity on fish scale surface.
2, the fish scale of cleaning is placed reactor, the water that adds 5000kg, add the stomach en-of 3kg, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 1.5,50 ℃ of hydrolysis temperatures, enzymolysis 6 hours, enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 20 minutes enzymes that go out, obtain marine organisms source protein matter.
3, after extracting solution is 10 μ m filter bag pre-treatment through the aperture, enter in the hold-up vessel of ceramic membrane equipment, utilizing the aperture is that 0.1 μ m ceramic membrane removes except macromole impurity, and film pressure is 0.3MPa, and temperature is 30 ℃.
4, sample utilizes relative molecular mass to remove small molecular weight impurity for the nanofiltration membrane of 2KD behind the purifying, and film pressure is 0.9MPa, and temperature is 25 ℃, obtains marine organisms source protein matter.
5, marine organisms source protein matter is packed in the reactor, add the fish scale fish-skin proteolytic enzyme of 2.7kg and the flavor protease of 0.3kg, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 7,50 ℃ of temperature of reaction were reacted 6 hours, and enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 30 minutes enzymes that go out, obtain marine organisms source step-down peptide solution.
6, utilize spray-drier rapid drying marine organisms source step-down peptide solution, air outlet temperature is that 70 ℃, inlet temperature are 140 ℃, spraying drying finishes, obtain marine organisms source step-down peptide powder finished product, the protein content of powder finished product is 91%, the averagemolecular wt amount is less than 4KD, and it is 54.9% that ACE suppresses activity.
Example two:
1, with the high-quality fish scale as raw material, selected fish scale can be that the marine fishes fish scale also can be the freshwater fish fish scale.Get fish scale 500kg and drop in the stirring and washing machine, add the water of 5000kg, clean 3 times, remove granule foreign.Fish scale after the washing is dropped in the cleaning machine, and adding 5000kg quality volumetric concentration is 5% sodium hydrogen carbonate solution, stirs 11 hours, washes.Adding the 5000kg concentration of volume percent is 5% sulphuric acid soln, stirs 8 hours, washes, and removes viscous substance, fat and other impurity on fish scale surface.
2, the fish scale of cleaning is placed reactor, the water that adds 5000kg, add the stomach en-of 1.5kg, utilize sodium hydroxide and salt acid for adjusting pH value, and control reaction system pH is 1,40 ℃ of hydrolysis temperatures, enzymolysis 6 hours, enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 20 minutes enzymes that go out, obtain marine organisms source protein matter.
3, after extracting solution is 10 μ m filter bag pre-treatment through the aperture, enter in the hold-up vessel of ceramic membrane equipment, utilizing the aperture is that 0.05 μ m ceramic membrane removes except macromole impurity, and film pressure is 0.3MPa, and temperature is 33 ℃.
4, sample utilizes relative molecular mass to remove small molecular weight impurity for the nanofiltration membrane of 300D behind the purifying, and film pressure is 0.9MPa, and temperature is 25 ℃, obtains marine organisms source protein matter.
5, marine organisms source protein matter is packed in the reactor, the papoid that adds 1.5kg, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 5.5,60 ℃ of temperature of reaction were reacted 5 hours, and enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 30 minutes enzymes that go out, obtain marine organisms source step-down peptide solution.
6, utilize spray-drier rapid drying marine organisms source step-down peptide solution, air outlet temperature is that 70 ℃, inlet temperature are 140 ℃, spraying drying finishes, obtain marine organisms source step-down peptide powder finished product, the protein content of powder finished product is 94%, the averagemolecular wt amount is less than 2KD, and it is 59.9% that ACE suppresses activity.
Example three:
1, with the high-quality fish scale as raw material, selected fish scale can be that the marine fishes fish scale also can be the freshwater fish fish scale.Get fish scale 500kg and drop in the stirring and washing machine, add the water of 5000kg, clean 3 times, remove granule foreign.Fish scale after the washing is dropped in the cleaning machine, and adding 5000kg quality volumetric concentration is 1% sodium hydroxide solution, stirs 11 hours, washes.Adding the 5000kg concentration of volume percent is 1% hydrochloric acid soln, stirs 9 hours, washes, and removes viscous substance, fat and other impurity on fish scale surface.
2, the fish scale of cleaning is placed reactor, the water that adds 5000kg, add the aspartic protease of 4.5kg, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 2.5,40 ℃ of hydrolysis temperatures, enzymolysis 3 hours, enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 20 minutes enzymes that go out, obtain marine organisms source protein matter.
3, after extracting solution is 10 μ m filter bag pre-treatment through the aperture, enter in the hold-up vessel of ceramic membrane equipment, utilizing the aperture is that 0.1 μ m ceramic membrane removes except macromole impurity, and film pressure is 0.3MPa, and temperature is 33 ℃.
4, sample utilizes relative molecular mass to remove small molecular weight impurity for the nanofiltration membrane of 2KD behind the purifying, and film pressure is 0.9MPa, and temperature is 25 ℃, obtains marine organisms source protein matter.
5, marine organisms source protein matter is packed in the reactor, the neutral protease that adds 4.5kg, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 7,50 ℃ of temperature of reaction were reacted 4 hours, and enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 30 minutes enzymes that go out, obtain marine organisms source step-down peptide solution.
6, utilize spray-drier rapid drying marine organisms source step-down peptide solution, air outlet temperature is that 70 ℃, inlet temperature are 140 ℃, spraying drying finishes, obtain marine organisms source step-down peptide powder finished product, the protein content of powder finished product is 90%, the averagemolecular wt amount is less than 4KD, and it is 74.5% that ACE suppresses activity.
Example four:
1, with the high-quality fish scale as raw material, selected fish scale can be that the marine fishes fish scale also can be the freshwater fish fish scale.Get fish scale 500kg and drop in the stirring and washing machine, add the water of 5000kg, clean 3 times, remove granule foreign.Fish scale after the washing is dropped in the cleaning machine, and adding 5000kg quality volumetric concentration is 1% sodium hydroxide solution, stirs 11 hours, washes.Adding the 5000kg concentration of volume percent is 1% sulphuric acid soln, stirs 9 hours, washes, and removes viscous substance, fat and other impurity on fish scale surface.
2, the fish scale of cleaning is placed reactor, the water that adds 5000kg, add the aspartic protease of 3kg, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 2.5,50 ℃ of hydrolysis temperatures, enzymolysis 6 hours, enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 20 minutes enzymes that go out, obtain marine organisms source protein matter.
3, after extracting solution is 10 μ m filter bag pre-treatment through the aperture, enter in the hold-up vessel of ceramic membrane equipment, utilizing the aperture is that 0.1 μ m ceramic membrane removes except macromole impurity, and film pressure is 0.3MPa, and temperature is 33 ℃.
4, sample utilizes relative molecular mass to remove small molecular weight impurity for the nanofiltration membrane of 2KD behind the purifying, and film pressure is 0.9MPa, and temperature is 25 ℃, obtains marine organisms source protein matter.
5, marine organisms source protein matter is packed in the reactor, the neutral protease and the 2.25kg bromeline that add 2.25kg, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 7,50 ℃ of temperature of reaction were reacted 4 hours, and enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 20 minutes enzymes that go out, obtain marine organisms source step-down peptide solution.
6, utilize spray-drier rapid drying marine organisms source step-down peptide solution, air outlet temperature is that 70 ℃, inlet temperature are 140 ℃, spraying drying finishes, obtain marine organisms source step-down peptide powder finished product, the protein content of powder finished product is 90%, the averagemolecular wt amount is less than 1KD, and it is 82.7% that ACE suppresses activity.
Example five:
1, with the high-quality fish scale as raw material, selected fish scale can be that the marine fishes fish scale also can be the freshwater fish fish scale.Get fish scale 500kg and drop in the stirring and washing machine, add the water of 5000kg, clean 3 times, remove granule foreign.Fish scale after the washing is dropped in the cleaning machine, and adding 5000kg quality volumetric concentration is 3% sodium hydrogen carbonate solution, stirs 10 hours, washes.Adding the 5000kg concentration of volume percent is 0.5% sulphuric acid soln, stirs 10 hours, washes, and removes viscous substance, fat and other impurity on fish scale surface.
2, the fish scale of cleaning is placed reactor, the water that adds 5000kg, the Sumizyme MP that adds 1.5kg utilizes sodium hydroxide to regulate the pH value, and control reaction system pH is 9,50 ℃ of hydrolysis temperatures, enzymolysis 6 hours, enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 20 minutes enzymes that go out, obtain marine organisms source protein matter.
3, after extracting solution is 1 μ m filter bag pre-treatment through the aperture, enter in the hold-up vessel of ceramic membrane equipment, utilizing the aperture is that 0.05 μ m ceramic membrane removes except macromole impurity, and film pressure is 0.3MPa, and temperature is 30 ℃.
4, sample utilizes relative molecular mass to remove small molecular weight impurity for the nanofiltration membrane of 1KD behind the purifying, and film pressure is 0.9MPa, and temperature is 28 ℃, obtains marine organisms source protein matter.
5, marine organisms source protein matter is packed in the reactor, the trypsinase that adds 3kg, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 8,40 ℃ of temperature of reaction were reacted 3 hours, and enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 30 minutes enzymes that go out, obtain marine organisms source step-down peptide solution.
6, utilize spray-drier rapid drying marine organisms source step-down peptide solution, air outlet temperature is that 80 ℃, inlet temperature are 150 ℃, spraying drying finishes, obtain marine organisms source step-down peptide powder finished product, the protein content of powder finished product is 92%, the averagemolecular wt amount is less than 5KD, and it is 67.4% that ACE suppresses activity.
Example six:
1, with the high-quality fish-skin as raw material, selected fish-skin can be that the marine fishes fish-skin also can be the freshwater fish fish-skin.Get fish-skin 500kg and drop in the stirring and washing machine, add the water of 5000kg, clean 3 times, remove granule foreign.Fish-skin after the washing is dropped in the cleaning machine, and adding 5000kg quality volumetric concentration is 5% sodium hydroxide solution, stirs 12 hours, washes.Adding the 5000kg concentration of volume percent is 2.5% hydrochloric acid soln, stirs 9 hours, washes, and removes viscous substance, fat and other impurity on fish scale surface.
2, the fish-skin of cleaning is placed reactor, the water that adds 5000kg, add the stomach en-of 4.5kg, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 1.5,35 ℃ of hydrolysis temperatures, enzymolysis 4 hours, enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 20 minutes enzymes that go out, obtain marine organisms source protein matter.
3, after extracting solution is 20 μ m filter bag pre-treatment through the aperture, enter in the hold-up vessel of ceramic membrane equipment, utilizing the aperture is that 0.1 μ m ceramic membrane removes except macromole impurity, and film pressure is 0.3MPa, and temperature is 35 ℃.
4, sample utilizes relative molecular mass to remove small molecular weight impurity for the nanofiltration membrane of 2KD behind the purifying, and film pressure is 0.9MPa, and temperature is 28 ℃, obtains marine organisms source protein matter.
5, marine organisms source protein matter is packed in the reactor, add 2.7kg sea-food proteolytic enzyme and 0.3kg flavor protease, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 7,50 ℃ of temperature of reaction were reacted 3 hours, and enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 30 minutes enzymes that go out, obtain marine organisms source step-down peptide solution.
6, utilize spray-drier rapid drying marine organisms source step-down peptide solution, air outlet temperature is that 80 ℃, inlet temperature are 150 ℃, spraying drying finishes, obtain marine organisms source step-down peptide powder finished product, the protein content of powder finished product is 91%, the averagemolecular wt amount is less than 5KD, and it is 72.6% that ACE suppresses activity.
Example seven:
1, with the high-quality fish-skin as raw material, selected fish-skin can be that the marine fishes fish-skin also can be the freshwater fish fish-skin.Get fish-skin 500kg and drop in the stirring and washing machine, add the water of 5000kg, clean 3 times, remove granule foreign.Fish-skin after the washing is dropped in the cleaning machine, and adding 5000kg quality volumetric concentration is 5% sodium hydroxide solution, stirs 10 hours, washes.Adding the 5000kg concentration of volume percent is 2.5% sulphuric acid soln, stirs 9 hours, washes, and removes viscous substance, fat and other impurity on fish scale surface.
2, the fish-skin of cleaning is placed reactor, the water that adds 5000kg, add the aspartic protease of 3kg, utilize sodium hydroxide and salt acid for adjusting pH value, and control reaction system pH is 2.5,50 ℃ of hydrolysis temperatures, enzymolysis 6 hours, enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 20 minutes enzymes that go out, obtain marine organisms source protein matter.
3, after extracting solution is 20 μ m filter bag pre-treatment through the aperture, enter in the hold-up vessel of ceramic membrane equipment, utilizing the aperture is that 0.05 μ m ceramic membrane removes except macromole impurity, and film pressure is 0.3MPa, and temperature is 35 ℃.
4, sample utilizes relative molecular mass to remove small molecular weight impurity for the nanofiltration membrane of 500D behind the purifying, and film pressure is 0.9MPa, and temperature is 28 ℃, obtains marine organisms source protein matter.
5, marine organisms source protein matter is packed in the reactor, add the 4.5kg bromeline, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 7,50 ℃ of temperature of reaction were reacted 4 hours, and enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 30 minutes enzymes that go out, obtain marine organisms source step-down peptide solution.
6, utilize spray-drier rapid drying marine organisms source step-down peptide solution, air outlet temperature is that 80 ℃, inlet temperature are 150 ℃, spraying drying finishes, obtain marine organisms source step-down peptide powder finished product, the protein content of powder finished product is 91%, the averagemolecular wt amount is less than 2KD, and it is 63.1% that ACE suppresses activity.
Example eight:
1, with the high-quality fish-skin as raw material, selected fish-skin can be that the marine fishes fish-skin also can be the freshwater fish fish-skin.Get fish-skin 500kg and drop in the stirring and washing machine, add the water of 5000kg, clean 3 times, remove granule foreign.Fish-skin after the washing is dropped in the cleaning machine, and adding 5000kg quality volumetric concentration is 4% sodium hydroxide solution, stirs 11 hours, washes.Adding the 5000kg concentration of volume percent is 0.5% hydrochloric acid soln, stirs 10 hours, washes, and removes viscous substance, fat and other impurity on fish scale surface.
2, the fish-skin of cleaning is placed reactor, the water that adds 5000kg, the Sumizyme MP that adds 3kg utilizes sodium hydroxide to regulate the pH value, and control reaction system pH is 9,40 ℃ of hydrolysis temperatures, enzymolysis 6 hours, enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 20 minutes enzymes that go out, obtain marine organisms source protein matter.
3, after extracting solution is 10 μ m filter bag pre-treatment through the aperture, enter in the hold-up vessel of ceramic membrane equipment, utilizing the aperture is that 0.05 μ m ceramic membrane removes except macromole impurity, and film pressure is 0.3MPa, and temperature is 35 ℃.
4, sample utilizes relative molecular mass to remove small molecular weight impurity for the nanofiltration membrane of 1KD behind the purifying, and film pressure is 0.9MPa, and temperature is 30 ℃, obtains marine organisms source protein matter.
5, marine organisms source protein matter is packed in the reactor, add 0.75kg trypsinase and 0.75kg Chymotrypsin, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 8,40 ℃ of temperature of reaction were reacted 3 hours, and enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 30 minutes enzymes that go out, obtain marine organisms source step-down peptide solution.
6, utilize spray-drier rapid drying marine organisms source step-down peptide solution, air outlet temperature is that 90 ℃, inlet temperature are 160 ℃, spraying drying finishes, obtain marine organisms source step-down peptide powder finished product, the protein content of powder finished product is 93%, the averagemolecular wt amount is less than 5KD, and it is 71.4% that ACE suppresses activity.
Example nine:
1, with the high-quality fish-skin as raw material, selected fish-skin can be that the marine fishes fish-skin also can be the freshwater fish fish-skin.Get fish-skin 500kg and drop in the stirring and washing machine, add the water of 5000kg, clean 3 times, remove granule foreign.Fish-skin after the washing is dropped in the cleaning machine, and adding 5000kg quality volumetric concentration is 4% sodium hydroxide solution, stirs 11 hours, washes.Adding the 5000kg concentration of volume percent is 0.5% sulphuric acid soln, stirs 10 hours, washes, and removes viscous substance, fat and other impurity on fish scale surface.
2, the fish-skin of cleaning is placed reactor, the water that adds 5000kg, the Sumizyme MP that adds 4.5kg utilizes sodium hydroxide to regulate the pH value, and control reaction system pH is 9,50 ℃ of hydrolysis temperatures, enzymolysis 6 hours, enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 20 minutes enzymes that go out, obtain marine organisms source protein matter.
3, after extracting solution is 10 μ m filter bag pre-treatment through the aperture, enter in the hold-up vessel of ceramic membrane equipment, utilizing the aperture is that 0.05 μ m ceramic membrane removes except macromole impurity, and film pressure is 0.3MPa, and temperature is 35 ℃.
4, sample utilizes relative molecular mass to remove small molecular weight impurity for the nanofiltration membrane of 1KD behind the purifying, and film pressure is 0.9MPa, and temperature is 30 ℃, obtains marine organisms source protein matter.
5, marine organisms source protein matter is packed in the reactor, add the 1.5kg Chymotrypsin, utilize sodium hydroxide and sulphur acid for adjusting pH value, and control reaction system pH is 8,40 ℃ of temperature of reaction were reacted 3 hours, and enzyme digestion reaction is warmed up to 80 ℃ after finishing, be incubated 30 minutes enzymes that go out, obtain marine organisms source step-down peptide solution.
6, utilize spray-drier rapid drying marine organisms source step-down peptide solution, air outlet temperature is that 90 ℃, inlet temperature are 160 ℃, spraying drying finishes, obtain marine organisms source step-down peptide powder finished product, the protein content of powder finished product is 93%, the averagemolecular wt amount is less than 5KD, and it is 68.8% that ACE suppresses activity.
Claims (9)
1. the preparation technology of a marine organisms source step-down peptide, it is characterized in that: it comprises the steps:
(1) pre-treatment
The marine organisms byproduct is dropped in the cleaning machine, add the alkaline solution of 10 times of marine organisms byproduct weight, the quality volumetric concentration of alkaline solution is 1-5%, stirs 10-12 hour, washes; Add the acid solution that is equivalent to 10 times of marine organisms byproduct weight again, the concentration of volume percent of acid solution is 0.5-5%, stirs 8-10 hour, and washing is clean, removes fat and other impurity of marine organisms byproduct; Described marine organisms byproduct is fish scale and fish-skin;
(2) extraction of marine organisms source protein matter
To place reactor through pre-treatment and the marine organisms byproduct of cleaning, add the water be equivalent to ten times of marine organisms byproduct weight and be equivalent to the feed intake proteolytic enzyme of weight 0.3-0.9% of marine organisms byproduct and carry out enzymolysis, hydrolysis temperature is 35-50 ℃, enzyme digestion reaction pH value is controlled at 1-9, enzymolysis 3-6 hour; Enzymolysis is warmed up to 80 ℃ after finishing, and is incubated 20 minutes enzymes that go out, and obtains marine organisms source protein matter; Used proteolytic enzyme is a kind of in stomach en-, aspartic protease, the Sumizyme MP;
(3) purifying of marine organisms source protein matter
Marine organisms source protein matter is placed ceramic membrane equipment, after filter bag is handled, carry out the micro-filtration removal of impurities, remove particulate contamination and the macromole impurity of sample; Get rid of small molecular weight impurity and inorganic salt with nanofiltration membrane again, obtain high-purity marine organisms source protein matter;
(4) preparation of marine organisms source step-down peptide
Add in high-purity marine organisms source protein matter and be equivalent to the feed intake proteolytic enzyme of weight 0.3-0.9% of albumen and carry out enzymolysis, hydrolysis temperature is 40-60 ℃, and enzyme digestion reaction pH value is controlled at 5-8, enzymolysis 3-6 hour; Enzymolysis is warmed up to 80 ℃ after finishing, and is incubated the 15-35 minute enzyme that goes out, and obtains marine organisms source step-down peptide solution; Used proteolytic enzyme is a kind of or two kinds of combinations in papoid, bromeline, neutral protease, trypsinase, Chymotrypsin, fish scale fish-skin proteolytic enzyme, sea-food proteolytic enzyme, the flavor protease;
(5) rapid drying
Utilize spraying drying rapid drying marine organisms source step-down peptide solution, obtain marine organisms source step-down peptide powder finished product.
2. the preparation technology of marine organisms source step-down peptide according to claim 1, it is characterized in that: the employed alkali of described step (1) pre-treatment is sodium bicarbonate or sodium hydroxide, and employed acid is sulfuric acid or hydrochloric acid.
3. the preparation technology of marine organisms source step-down peptide according to claim 1 is characterized in that: regulating pH alkali in the described step (2) is sodium hydroxide; Regulating pH acid is sulfuric acid or hydrochloric acid.
4. the preparation technology of marine organisms source step-down peptide according to claim 1, it is characterized in that: the aperture of employed filter bag is 1-20 μ m in the described step (3).
5. the preparation technology of marine organisms source step-down peptide according to claim 1, it is characterized in that: the aperture of employed ceramic membrane is 0.05-0.1 μ m in the described step (3), and film pressure is 0.3MPa, and temperature is 30-35 ℃.
6. the preparation technology of marine organisms source step-down peptide according to claim 1, it is characterized in that: employed nanofiltration membrane relative molecular mass is 300D-2kD in the described step (3), and film pressure is 0.9MPa, and temperature is 25-30 ℃.
7. the preparation technology of marine organisms source step-down peptide according to claim 1 is characterized in that: regulating pH alkali in the described step (4) is sodium hydroxide; Regulating pH acid is sulfuric acid.
8. the preparation technology of marine organisms source step-down peptide according to claim 1 is characterized in that: spray-dired air outlet temperature is that 70-90 ℃, inlet temperature are 140-180 ℃ in the described step (5).
9. the preparation technology of marine organisms source step-down peptide according to claim 1 is characterized in that: before step (1) pre-treatment step, earlier the marine organisms byproduct is cleaned to remove granule foreign.
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| CN103305578B (en) * | 2013-06-03 | 2015-01-07 | 陈锦权 | Method for preparing hypotensive substance by using abalone visceral connective tissues |
| CN103388017B (en) * | 2013-08-06 | 2014-12-17 | 国家海洋局第三海洋研究所 | Preparation technology of fish skin collagen oligopeptide |
| CN103980347B (en) * | 2014-05-22 | 2017-05-17 | 浙江海洋学院 | Antihypertensive peptide of swim bladder of large yellow croaker and preparation method and use thereof |
| CN104031967B (en) * | 2014-06-17 | 2016-08-24 | 暨南大学 | A kind of sardine blood pressure lowering peptide and preparation method and application |
| CN105821108A (en) * | 2016-05-06 | 2016-08-03 | 浙江万里学院 | Method for preparing Chinese softshell turtle antihypertensive peptide by means of mixed enzymolysis and film filtration |
| CN106350560B (en) * | 2016-11-08 | 2020-07-03 | 广东海洋大学 | Preparation method of fish protein peptide, fish protein peptide obtained by preparation method and application of fish protein peptide |
| CN106834399A (en) * | 2016-12-30 | 2017-06-13 | 浙江海洋大学 | A kind of Antihypertensive Peptides from Trachyostracous mussel closed shell flesh |
| CN107440010A (en) * | 2017-06-22 | 2017-12-08 | 新昌县南翔食品科技有限公司 | Compound based on sardine activity extract and preparation method thereof |
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