CN104975059A - Method for preparing ACE (angiotensin converting enzyme) inhibitory peptide by using cod meat - Google Patents
Method for preparing ACE (angiotensin converting enzyme) inhibitory peptide by using cod meat Download PDFInfo
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- CN104975059A CN104975059A CN201410138184.8A CN201410138184A CN104975059A CN 104975059 A CN104975059 A CN 104975059A CN 201410138184 A CN201410138184 A CN 201410138184A CN 104975059 A CN104975059 A CN 104975059A
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Abstract
The invention discloses a method for preparing an ACE (angiotensin converting enzyme) inhibitory peptide by using cod meat. The method comprises the following steps: cleaning the cod meat, mixing the cod meat with water, homogenizing, performing enzymolysis, performing plate-frame filtration, performing ultrafiltration to withhold small molecules of less than 3000Da, and drying to obtain the ACE inhibitory peptide. Further, the ACE inhibitory peptide is subjected to two-step separation and purification by using Sephadex G-15 and RP-HPLC to obtain the high-purity ACE inhibitory peptide. By using the method disclosed by the invention, the low-value cod meat can be converted into the ACE inhibitory peptide with relatively high values, so that the waste is changed into valuable, resources are fully used, and the method has the advantages that the process route is simple and feasible, the operation is simple, the used equipment is mature equipment, the investment cost is relatively low, and the recovery rate of the ACE inhibitory peptide is high.
Description
Technical field
The present invention relates to food field of deep, particularly the deep processing of the cod flesh of fish.
Background technology
Hypertension as a kind of multi-pathogenesis, progressivity cardiovascular disorder, the initiatively elevation of blood pressure of clinical manifestation whole body, the systolic pressure >=140mmHg of patient, diastolic pressure >=90mmHg.Along with the development of the state of an illness, blood pressure continues to raise, and then occurs the organ damages such as the heart, brain, kidney and retina, causes congestive heart failure, cerebral apoplexy, coronary heart disease, renal failure, the sickness rate of aortic aneurysm and case fatality rate to raise.Hypertension is the high disease of a kind of morbidity, has become the great public health problem in global range.According to the press Communique that International Society of Hypertension delivers, global hypertension or existing 9.72 hundred million people of slight Hypertension crowd, account for world adult population's 26.4%.The World Health Organization (WHO) to the necrology of the various disease in the whole world, the ratio that the cardiovascular and cerebrovascular diseases death toll being representative with hypertension etc. accounts for total death toll by by 1997 28.8% rise to 2002 36.0%.China's hypertensive epidemiological survey result display,
Medicine at present for hypertension therapeutic mainly contains six large classes: be diuretic antihypertensive medicine, beta-blocker, calcium antagonist, angiotensin-convertion enzyme inhibitor (ACEI), angiotensin II receptor antagonists (ARB) and alpha 1-receptor retarding agent respectively.These medicines achieve good effect in hypertensive treatment.Wherein angiotensin-convertion enzyme inhibitor is the line antihypertensive drug uniquely having whole 6 powerful adaptations (heart failure, myocardial infarction, high risk factors, diabetes, chronic nephropathy, prevention of recurrence of stroke).ACEI obtains certainly renal hypertension, chronic glomerulonephritis, chronic renal failure and using of Prevention diabetic nephropathy.
Ace inhibitory peptide refers to the polypeptide that a class has ACE inhibitory activity.Aminoacid sequence and the peptide chain length of these polypeptide are had nothing in common with each other, but all have similar activity.ACE has two reactive sites, respectively N-district by name, C-district, and these two active regions have similar function, just different to the avidity of different substrate.Ace inhibitory peptide to the combination of ACE, killer tube Angiotensin Converting Enzyme I, thus hinder the hydrolysis of ACE catalysis angiotensin I to become Angiotensin II, thus play hypotensive activity.At present, much research shows that some small peptides in food exist hypotensive activity.People have found ace inhibitory peptide from the material in numerous food product source.From source, food source ACE inhibitor peptides can be divided three classes: from the peptide class of milk-protein, comprise milk casein, lactoglobulin and whey-protein; From the peptide class of vegetable-protein, as large mpd polypeptide, soybean polypeptide, Semen Maydis polypeptide etc.; From the peptide class of fish and shellfish, as tilapia polypeptide, hairtail polypeptides, tuna polypeptide, sardines polypeptide, krill polypeptide etc.Although the antihypertensive effect of these ace inhibitory peptides does not have synthetic drugs strong, they have safely, the feature that has no side effect has attracted numerous investigator.The short chain ace inhibitory peptide found from whole food albumen, not only experiment in vitro proves there is stronger inhibit activities to ACE, and the blood pressure lowering effect of experimentation on animals and human clinical trial is also very exciting.This is for prevent and to alleviate hypertension to provide new approach by functional food.
Cod is mainly distributed in North Pacific, both sides, the North Atlantic generally dwell in near-bottom, take pelagic realm to by offshore, be mainly distributed in Bohai and Yellow Seas and northern East China Sea in China.There is main fishing ground in Northern Huanghai Sea, Shandong angle of elevation southeast by east and oceanic island south and sea area, the southeast.Cod meat is white carefully fresh and tender, tasty and refreshing oiliness, in the world many countries using cod as main edible fishes.
Summary of the invention
The object of the present invention is to provide a kind of method using the cod flesh of fish to prepare ace inhibitory peptide.
The technical solution used in the present invention is:
Utilize cod to oppress the method manufacturing ace inhibitory peptide, comprise the steps:
1) cleaned by the cod flesh of fish, add water, the post-heating of homogenized, by enzyme-deactivating, obtains slurries A;
2) slurries A is cooled to not higher than 60 DEG C, adds protease hydrolyzed, obtain enzymolysis solution;
3) enzymolysis solution is centrifugal, collect supernatant A;
4) in supernatant A, add calcium carbonate, mixing, filter the supernatant liquor B obtaining clarifying;
5) use the hollow-fibre ultrafiltration device ultrafiltration of molecular weight cut-off 3000Da, collect filtrate, drying obtains ace inhibitory peptide.
As further optimization of the present invention, by supernatant A heating to make proteolytic enzyme deactivation wherein.
As further optimization of the present invention, the ace inhibitory peptide that drying obtains obtains highly purified ace inhibitory peptide further across the separation and purification of Sephadex G-15 and RP-HPLC two step.
As further optimization of the present invention, be heated to 90 DEG C after homogenized and be incubated at least 10min, with by enzyme-deactivating.
As further optimization of the present invention, slurries A is cooled to 45 ~ 55 DEG C, regulates its pH to 7.5 ~ 8.5, add protease hydrolyzed afterwards.
As further optimization of the present invention, the cod flesh of fish is 1: 3 with the mass ratio of water.
As further optimization of the present invention, the add-on of calcium carbonate is 10% of supernatant A quality.
As further optimization of the present invention, the enzymolysis time of proteolytic enzyme is 120 ~ 240min.
The invention has the beneficial effects as follows:
The cod of the low value flesh of fish can be converted into the ace inhibitory peptide with higher-value by the inventive method, turns waste into wealth, takes full advantage of resource.
The inventive method operational path is simple, simple to operate, and the equipment used is mature equipment, and cost of investment is lower, and the ace inhibitory peptide rate of recovery is high.
Obtain highly purified ace inhibitory peptide through the separation and purification of Sephadex G-15 and RP-HPLC two step, have fabulous biological activity, it suppresses the IC of ACE
50value is 10.7 μMs.
Embodiment
Utilize cod to oppress the method manufacturing ace inhibitory peptide, comprise the steps:
1) cleaned by the cod flesh of fish, add water, the post-heating of homogenized, by enzyme-deactivating, obtains slurries A;
2) slurries A is cooled to not higher than 60 DEG C, adds protease hydrolyzed, obtain enzymolysis solution;
3) enzymolysis solution is centrifugal, collect supernatant A;
4) in supernatant A, add calcium carbonate, mixing, filter the supernatant liquor B obtaining clarifying;
5) use the hollow-fibre ultrafiltration device ultrafiltration of molecular weight cut-off 3000Da, collect filtrate, drying obtains ace inhibitory peptide.
As further optimization of the present invention, by supernatant A heating to make proteolytic enzyme deactivation wherein.Certainly, also as required, can be used it. his known method is by enzyme-deactivating wherein.
As further optimization of the present invention, the ace inhibitory peptide that drying obtains obtains highly purified ace inhibitory peptide further across the separation and purification of Sephadex G-15 and RP-HPLC two step.
As further optimization of the present invention, be heated to 90 DEG C after homogenized and be incubated at least 10min, with by enzyme-deactivating.Certainly, also can as required, use other known methods by enzyme-deactivating wherein.
As further optimization of the present invention, slurries A is cooled to 45 ~ 55 DEG C, regulates its pH to 7.5 ~ 8.5, add protease hydrolyzed afterwards.
As further optimization of the present invention, the cod flesh of fish mixes with water, is conveniently homogenate damage, its mass ratio can adjust as required, especially, when its mass ratio is 1: 3, effectively can reduce the consumption of water, meanwhile, also smaller to the preparation disadvantageous effect of peptide.
As further optimization of the present invention, the add-on of calcium carbonate is 10% of supernatant A quality.
As further optimization of the present invention, the enzymolysis time of proteolytic enzyme is 120 ~ 240min.Certainly, according to the difference of used enzyme and hydrolysis temperature, also suitable shortening or prolongation can be carried out.
Below in conjunction with embodiment, further illustrate the present invention.
The enzymolysis of the embodiment 1 cod flesh of fish and the enrichment of ace inhibitory peptide
1) process of raw material: take the 100kg cod flesh of fish, after wash clean, add 300L deionized water, after carrying out making beating process, boils 20min, deactivation self enzyme system in hollander;
2) the enzymolysis cod flesh of fish: treat cod flesh of fish slurries, be cooled to 55 DEG C, adjust pH to 8.0, add into Sumizyme MP Alcalase150g, enzymolysis 3hr, obtains enzymolysis solution;
3) deactivation Sumizyme MP Alcalase: enzymolysis solution is warming up to 95 DEG C, insulation 10min, the Sumizyme MP Alcalase in deactivation enzymolysis solution;
4) pre-treatment of enzymolysis solution: the enzymolysis solution after the enzyme that goes out is cooled to room temperature, adopts tubular-bowl centrifuge, centrifugal under 3000rpm, collects supernatant liquor;
5) clarification of enzymolysis solution: add 10% light calcium carbonate in the supernatant liquor of collected by centrifugation, obtains after Plate Filtration clarifying, containing the clear liquid of ace inhibitory peptide;
6) removal of macromolecular substance: the hollow-fibre ultrafiltration device adopting molecular weight cut-off 3000Da, ultrafiltration is removed containing the macromolecular substance in the clear liquid of ace inhibitory peptide, collects the filtrate containing ace inhibitory peptide;
7) drying of ace inhibitory peptide: by filtrate spraying dry, namely obtain ace inhibitory peptide.
The preparation of embodiment 2 high purity ace inhibitory peptide
Sephadex G-15 is adopted to be separated the ace inhibitory peptide of crude product ace inhibitory peptide powder, gel column, 1.8 × 60cm; Sample concentration, 50mg/ml; Applied sample amount, 1.5ml; Flow velocity, 0.4ml/min; Eluent, 0.02mol/L HAc-NaAc damping fluid (pH4.0); Obtain 7 components, wherein the activity of component E is higher;
Table 1Sephadex G-15 is separated the ACE inhibitory activity of enzymolysis product obtained component
RP-HPLC is utilized to carry out purifying to component E, use Kromasil C18 12 μm of ST4.6/250 (4.6 × 250mm) chromatographic columns to be separated and obtain 16 components, wherein component F10 has higher ACE inhibitory activity, carry out through amino acid composition analysis and qualification to the single-activity component F10 that purifying obtains, a kind of new ace inhibitory peptide LPP, its IC is isolated from cod flesh of fish Alcalase zymolyte
50value is 10.7 μMs.
Claims (9)
1. utilize cod to oppress the method manufacturing ace inhibitory peptide, comprise the steps:
1) cleaned by the cod flesh of fish, add water, the post-heating of homogenized, by enzyme-deactivating, obtains slurries A;
2) slurries A is cooled to not higher than 60 DEG C, adds protease hydrolyzed, obtain enzymolysis solution;
3) enzymolysis solution is centrifugal, collect supernatant A;
4) in supernatant A, add calcium carbonate, mixing, filter the supernatant liquor B obtaining clarifying;
5) use the hollow-fibre ultrafiltration device ultrafiltration of molecular weight cut-off 3000Da, collect filtrate, drying obtains ace inhibitory peptide.
2. method according to claim 1, is characterized in that: by supernatant A heating to make proteolytic enzyme deactivation wherein.
3. method according to claim 1 and 2, is characterized in that: be heated to 90 DEG C after homogenized and be incubated at least 10min, with by enzyme-deactivating.
4. the method according to claims 1 to 3 any one, is characterized in that: slurries A is cooled to 45 ~ 55 DEG C, regulates its pH to 7.5 ~ 8.5, add protease hydrolyzed afterwards.
5. the method according to Claims 1 to 4 any one, is characterized in that: the cod flesh of fish is 1: 3 with the mass ratio of water.
6. the method according to Claims 1 to 5 any one, is characterized in that: proteolytic enzyme is Sumizyme MP Alcalase.
7. the method according to claim 1 ~ 6 any one, is characterized in that: the add-on of calcium carbonate is 10% of supernatant A quality.
8. the method according to claim 1 ~ 7 any one, is characterized in that: the enzymolysis time of proteolytic enzyme is 120 ~ 240min.
9. the method according to claim 1 ~ 8 any one, is characterized in that: the ace inhibitory peptide that drying obtains obtains highly purified ace inhibitory peptide further across the separation and purification of Sephadex G-15 and RP-HPLC two step.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355344A (en) * | 2019-01-08 | 2019-02-19 | 中国科学院烟台海岸带研究所 | A kind of preparation method of antihypertensive activity peptide |
| CN110521849A (en) * | 2019-08-26 | 2019-12-03 | 大连工业大学 | A kind of preparation method and applications with the high gadus protein peptides for digesting and assimilating characteristic |
| CN113005166A (en) * | 2021-04-14 | 2021-06-22 | 中国海洋大学 | Cod polypeptide with xanthine oxidase inhibitory activity |
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| CN101240312A (en) * | 2008-01-21 | 2008-08-13 | 南昌大学 | Method for preparing ACE inhibition peptide originate from fish skin |
| CN102586374A (en) * | 2012-01-17 | 2012-07-18 | 广西大学 | Angiotensin-converting enzyme inhibitory peptide and preparation method thereof |
| CN102808010A (en) * | 2011-05-30 | 2012-12-05 | 浙江海洋学院 | Method for preparing antihypertensive peptides through enzymolysis of ground meat proteins of tuna |
| CN103571904A (en) * | 2013-10-15 | 2014-02-12 | 江苏科技大学 | Method for preparing channel catfish skin collagen ACE (angiotensin-1 converting enzyme) inhibitory peptides |
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2014
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Patent Citations (4)
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|---|---|---|---|---|
| CN101240312A (en) * | 2008-01-21 | 2008-08-13 | 南昌大学 | Method for preparing ACE inhibition peptide originate from fish skin |
| CN102808010A (en) * | 2011-05-30 | 2012-12-05 | 浙江海洋学院 | Method for preparing antihypertensive peptides through enzymolysis of ground meat proteins of tuna |
| CN102586374A (en) * | 2012-01-17 | 2012-07-18 | 广西大学 | Angiotensin-converting enzyme inhibitory peptide and preparation method thereof |
| CN103571904A (en) * | 2013-10-15 | 2014-02-12 | 江苏科技大学 | Method for preparing channel catfish skin collagen ACE (angiotensin-1 converting enzyme) inhibitory peptides |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355344A (en) * | 2019-01-08 | 2019-02-19 | 中国科学院烟台海岸带研究所 | A kind of preparation method of antihypertensive activity peptide |
| CN110521849A (en) * | 2019-08-26 | 2019-12-03 | 大连工业大学 | A kind of preparation method and applications with the high gadus protein peptides for digesting and assimilating characteristic |
| CN113005166A (en) * | 2021-04-14 | 2021-06-22 | 中国海洋大学 | Cod polypeptide with xanthine oxidase inhibitory activity |
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