CN104592350A - ACE (angiotension converting enzyme) inhibitory peptide and preparation method thereof - Google Patents
ACE (angiotension converting enzyme) inhibitory peptide and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an ACE (angiotension converting enzyme) inhibitory peptide and a preparation method thereof, and provides an ACE inhibitory peptide with an anti-hypertension effect and a preparation method of the ACE inhibitory peptide. An amino acid sequence of the ACE inhibitory peptide is YS. The preparation method comprises the following steps: sufficiently mixing the ACE inhibitory peptide with a milk powder substrate to prepare anti-hypertension milk powder; or mixing the ACE inhibitory peptide with fresh milk, and inoculating lactic acid bacteria to ferment to obtain yoghourt with the anti-hypertension effect. The milk product has a remarkable antihypertensive effect, is free of interferences on normal blood pressure, safe to eat, and suitable for the hypertension crowds.
Description
The application is application number is 201310239898.3, and the applying date is 2013-06-18, and denomination of invention is the divisional application of " a kind of ace inhibitory peptide and preparation method thereof ".
Technical field
The present invention relates to biological technical field, particularly relating to one is take bovine casein as prepared using stomach en-and trypsin hydrolyzing, and preparation has the peptide of ACE inhibitory activity, and is added in milk powder matrix, produces the milk powder with antihypertensive function; Or add in the fresh milk of sterilization, the Yoghourt with antihypertensive function is obtained through lactic fermentation.
Background technology
Along with the development in the world and the progress of society, the standard of living of people improves constantly; Popular mode of life and dietary structure all there occurs great variety, and modern civilization diseases is also comed one after another.
Modern civilization diseases mainly comprises obesity, diabetes, hypertension, dysthymia disorders etc.In these common diseases, hypertension is one of chronic disease of serious harm human health, is also worldwide common condition, its reason occurred, and it be not immediately clear; Meanwhile, hypertension is controlled risk factor main in cardiovascular disorder evolution, often reduces 5mmHg systolic pressure, and the risk suffering from cardiovascular disorder just reduces by 16%; The blood pressure continuing to raise increases the risk of apoplexy, heart attack and renal failure most probably, and every year, there is the grownup of about 30% or more in the whole world by the puzzlement of high blood pressure disease, and has millions of people dead because of hypertension and complication thereof.Hypertension has become one of principal disease jeopardizing human body health, and according to World Health Organization's prediction, to the year two thousand twenty, Non Communicable Diseases (NCD) will account for 79% of the cause of death, and wherein the cardiovascular disorder such as hypertension will account for first place.As the hypertension of one of modern civilization diseases mark, be usually called the disease of " without omen " by people.That be the consequence caused due to hypertension wait until always significantly infringement is produced to body after just realized, at this moment often too late.Therefore, the prevention and therapy work of high blood pressure disease is particularly important.
During hypertension incidence, needs of patients often takes some secondary buck medicines, but these depressor can produce many side effects, especially very large to renal function.So, if some food containing hypertension composition can be eaten in diet, so not only can effectively control hypertensive frequency of disease development, and without any side effects to health.
Biologically active peptides is the peptide class general name with physiological regulation function, and generally speaking, the most relative molecular mass of this kind of peptide is less, in human body to digest and assimilate comparatively protein easy.These little peptides can not only provide growth in humans to grow required nutrition, can also regulate human physiological functions, play the effect of prevention, even disease therapy.The kind of food source property biologically active peptides is more, comprise angiotensin-converting enzyme (angiotensionconverting enzyme, ACE) inhibiting peptide, immunomodulatory peptides, anti-oxidation peptide, antibacterial peptide, antithrombotic peptide, opioid peptides, calcium binding peptide.The wherein prevention and therapy close relation of ace inhibitory peptide and high blood pressure disease, has caused showing great attention to of various countries scientist and government.
In food source property biologically active peptides, remarkable with the effect of milk-derived biologically active peptides again.It refers to some fragment of some protein peptide chain of Ruzhong same or similar, in Ruzhong intrinsic or produce in milk-protein degradation process there is bioactive peptide class.Along with the discovery of the milk-derived bioactive peptide of difference in functionality, milk-protein Quality Research is become to the study hotspot of physiology educational circles and nutrition educational circles.
Casein (casein) is the most rich in protein of Ruzhong content, in its macromole, containing multiple, there is bioactive small molecule segment, under the effect of different proteolytic enzyme, can discharge the molecule fragment with difference in functionality, ace inhibitory peptide is exactly one wherein.Recent domestic result of study shows, when Ruzhong casein is in vivo or when using enzymic digestion in food processing process, can produce a large amount of ace inhibitory peptide, these ace inhibitory peptides have have no side effect, the advantage such as security is high, easy absorption.
The ace inhibitory peptide of current report comprises: the α that Maruyama and Suzuki exists in the tryptic hydrolysates of nineteen eighty-two report bovine casein
s1-caseic f23 ~ 24, f23 ~ 27 and f174 ~ 199; F177 ~ 183 of beta-casein, f193 ~ 202 fragment.As.1398 neutral proteinase hydrolysis bovine casein gained two peptide sections such as Jiang, i.e. Arg-Tyr-Pro-Ser-Tyr-Gly (κ casein, f (25 ~ 30)) and Asp-Glu-Arg-Phe (κ casein, f (15 ~ 18)), and record its IC
50value is respectively (54 ± 1.2) μ g/mL and (21 ± 0.8) μ g/mL.
Summary of the invention
The object of the invention is the technological deficiency for existing in prior art, and provide a kind of there is antihypertensive function, the casein hydrolysate being rich in ace inhibitory peptide and preparation method thereof.
Another object of the present invention is to provide a kind of ace inhibitory peptide with antihypertensive function and preparation method thereof.
Another object of the present invention is to provide a kind of milk powder and the Yoghourt with antihypertensive function.
Another object of the present invention is to provide a kind of preparation method with the Yoghourt of antihypertensive function.
The technical scheme adopted for realizing object of the present invention is:
Be rich in a casein hydrolysate for ace inhibitory peptide, have the peptide fragment of ACE inhibitory activity containing two, the aminoacid sequence of the peptide fragment of two described ACE inhibitory activity is respectively: YS and YQKFPQYLQY.
A kind of ace inhibitory peptide, its aminoacid sequence is: YS.
A kind of ace inhibitory peptide, its aminoacid sequence is: YQKFPQYLQY.
Be rich in a preparation method for the casein hydrolysate of ace inhibitory peptide, it is characterized in that, comprise the steps:
(1) at concentration of substrate be 7% caseic aqueous solution in, the ratio being 1:100 according to the ratio of enzyme-to-substrate adds stomach en-, and pepsic hydrolysising condition is: temperature is 37 DEG C, pH value is 2.0-3.0, after hydrolysis 3h, terminate reaction with boiling water bath heating 10min, be cooled to room temperature;
(2) pH value of set-up procedure (1) gained hydrolyzed solution is to 7.7-8.5, then continues hydrolysis with trypsinase, and tryptic hydrolysising condition is: temperature is 45-50 DEG C, and pH value is 7.7-8.5, and the ratio of Trypsin enzyme-to-substrate is 1:500; After hydrolysis 3h, hydrolyzed solution boils 10min with boiling water bath and to go out enzyme, is then cooled to room temperature; The pH value of regulation system is 6.8-7.0; Afterwards, centrifugal, get supernatant liquor;
(3) step (2) gained supernatant liquor is carried out uf processing, molecular weight cut-off is 6Ku, collect ultrafiltration and appear thing, carry out vacuum-drying, obtain the hydrolysis ultrafiltration thing that moisture is less than 5%, obtain the casein hydrolysate being rich in ace inhibitory peptide, wherein have ACE inhibitory activity containing two fragments, aminoacid sequence is respectively YS and YQKFPQYLQY.
A kind of preparation method being rich in the casein hydrolysate of ace inhibitory peptide, flavor protease debitterize is added after trypsin hydrolyzing, the hydrolysising condition of flavor protease is: the ratio of flavor protease and substrate is 3200U/g, and hydrolysis temperature is 45-50 DEG C, pH value is 6.8-7.0, hydrolysis time is 1.5-2 hour.
A preparation method for ace inhibitory peptide, comprises the steps:
(1) at concentration of substrate be 7% caseic aqueous solution in, the ratio being 1:100 according to the ratio of enzyme-to-substrate adds stomach en-, pepsic hydrolysising condition is: temperature is 37 DEG C, pH value is 2.0-3.0, after hydrolysis 3h, terminate reaction with boiling water bath heating 10min, be cooled to 45-50 DEG C;
(2) pH value of set-up procedure (1) gained hydrolyzed solution is to 7.7-8.5, then continues hydrolysis with trypsinase, and tryptic hydrolysising condition is: temperature is 45-50 DEG C, and pH value is 7.7-8.5, and the ratio of Trypsin enzyme-to-substrate is 1:500; After hydrolysis 3h, hydrolyzed solution boils 10min with boiling water bath and to go out enzyme, is then cooled to room temperature; The pH value of regulation system is back to 7.0; Afterwards, centrifugal, get supernatant liquor;
(3) step (2) gained supernatant liquor is carried out uf processing, molecular weight cut-off is 6Ku, collects ultrafiltration and appears thing;
(4) ultrafiltration is appeared thing and is separated by Sephadex G-15, obtains three components, detects the ACE inhibitory activity of three components respectively;
(5) select the high component chromatographic mass spectrometry detecting instrument of the middle ACE inhibitory activity of step (4) to carry out the mass spectrometric detection of aminoacid sequence, obtaining aminoacid sequence is the fragment of YS; Adopt solid phase synthesis technique to synthesize, obtain the bioactive peptide with ACE inhibit feature.
A preparation method for ace inhibitory peptide, comprises the steps:
(1) at concentration of substrate be 7% caseic aqueous solution in, the ratio being 1:100 according to the ratio of enzyme-to-substrate adds stomach en-, pepsic hydrolysising condition is: temperature is 37 DEG C, pH value is 2.0-3.0, after hydrolysis 3h, terminate reaction with boiling water bath heating 10min, be cooled to 45-50 DEG C;
(2) pH value of set-up procedure (1) gained hydrolyzed solution is to 7.7-8.5, then continues hydrolysis with trypsinase, and tryptic hydrolysising condition is: temperature is 45-50 DEG C, and pH value is 7.7-8.5, and the ratio of Trypsin enzyme-to-substrate is 1:500; After hydrolysis 3h, hydrolyzed solution boils 10min with boiling water bath and to go out enzyme, is then cooled to room temperature; The pH value of regulation system is back to 7.0; Afterwards, centrifugal, get supernatant liquor;
(3) step (2) gained supernatant liquor is carried out uf processing, molecular weight cut-off is 6Ku, collects ultrafiltration and appears thing;
(4) ultrafiltration is appeared thing and is separated by Sephadex G-15, obtains three components, detects the ACE inhibitory activity of three components respectively;
(5) select the high component chromatographic mass spectrometry detecting instrument of the middle ACE inhibitory activity of step (4) to carry out the mass spectrometric detection of aminoacid sequence, obtaining aminoacid sequence is the fragment of YQKFPQYLQY; Adopt solid phase synthesis technique to synthesize, obtain the bioactive peptide with ACE inhibit feature.
A kind of antihypertensive milk powder being rich in ace inhibitory peptide, add the casein hydrolysate of claim 4 gained in milk powder matrix, or to add aminoacid sequence that claim 6 obtains in milk powder matrix be the ace inhibitory peptide of YS and/or the aminoacid sequence of claim 7 gained is the ace inhibitory peptide of YQKFPQYLQY.
Preferably add the casein hydrolysate of 12.6 grams of claim 4 gained in 100 grams of milk powder matrix, or add ace inhibitory peptide that 0.6 gram of aminoacid sequence is YS in 100 grams of milk powder matrix and/or aminoacid sequence is the ace inhibitory peptide of YQKFPQYLQY, when to add ace inhibitory peptide that the aminoacid sequence that obtains is YS and aminoacid sequence be the ace inhibitory peptide of YQKFPQYLQY, two kinds of ace inhibitory peptides can add according to arbitrary proportion mixing.Two kinds of ace inhibitory peptides preferably add according to the ratio of 1:1.
A kind of antihypertensive Yoghourt being rich in ace inhibitory peptide, add the casein hydrolysate of claim 4 gained in fresh milk, or to add aminoacid sequence that claim 6 obtains in fresh milk be the ace inhibitory peptide of YS and/or the aminoacid sequence of claim 7 gained is the ace inhibitory peptide of YQKFPQYLQY.
Preferably add the casein hydrolysate of 1.26 grams of claim 4 gained in every 100 ml fresh milks, or 100 add ace inhibitory peptide that 60 milligrams of aminoacid sequences are YS in ml fresh milk and/or aminoacid sequence is the ace inhibitory peptide of YQKFPQYLQY, when to add ace inhibitory peptide that the aminoacid sequence that obtains is YS and aminoacid sequence be the ace inhibitory peptide of YQKFPQYLQY, the two carries out mixing rear interpolation according to arbitrary proportion.Two kinds of ace inhibitory peptides preferably add according to the ratio of 1:1.
A preparation method for antihypertensive Yoghourt, comprises the steps:
(1) in the fresh milk through sterilization, add the casein hydrolysate being rich in ace inhibitory peptide prepared in claim 4, fully mix; Or in the fresh milk through sterilization, to add aminoacid sequence be the ace inhibitory peptide of YS and/or aminoacid sequence is the ace inhibitory peptide of YQKFPQYLQY, fully mixes;
(2) inoculate 0.06% and deliver directly milk-acid bacteria, 42 DEG C ferment 5 hours, obtain the Yoghourt with antihypertensive function.
Compared with prior art, the invention has the beneficial effects as follows:
1, two peptide fragment containing ACE inhibit feature in casein hydrolysate of the present invention, correspond to N end: α by analysis
2f (56-57): YS and α
2f (98-107): YQKFPQYLQY, be a kind of novel bioactive peptide with ACE inhibit feature, after testing there is antihypertensive functional.
2, the aminoacid sequence of bioactive peptide of the present invention is respectively YS and YQKFPQYLQY, is a kind of novel bioactive peptide with ACE inhibit feature, has antihypertensive functional after testing.
3, with the peptide fragment with ACE activity inhibition coming from bovine casein obtained, [N holds milk-product of the present invention: α
2f (56-57): YS, α
2be main functional component, the milk powder with antihypertensive function produced, compared with milk powder matrix, has remarkable antihypertensive function (p < 0.05) f (98-107): YQKFPQYLQY]; The Yoghourt of the antihypertensive function produced, compared with common fermentation Yoghourt, has remarkable antihypertensive function (p < 0.05).And on normal arterial pressure without impact, edible safety, is applicable to Hypertensive Population and eats.
4, the raw material producing ace inhibitory peptide in the present invention is bovine casein, and produce stomach en-existing for digestion of ace inhibitory peptide lytic enzyme used and trypsinase, the main raw material of product is milk powder and milk.Therefore, the product edible safety of production.
5, the preparation method of casein hydrolysate of the present invention and ace inhibitory peptide is simple, practical.
Accompanying drawing explanation
Figure 1 shows that the ACE inhibiting rate of fragment YQK FPQ YLQY;
Figure 2 shows that fragment α
2the ACE inhibition of f (56-57): YS;
Figure 3 shows that gavage fragment α
2f (56-57): YS is on the impact of SHR blood pressure;
Figure 4 shows that gavage fragment α
2f (98-107): YQKFPQYLQY is on the impact of SHR blood pressure;
Figure 5 shows that gavage fragment α
2f (56-57): YS is on the impact of wistar rat blood pressure;
Figure 6 shows that gavage fragment α
2f (98-107): YQKFPQYLQY is on the impact of wistar rat blood pressure;
Figure 7 shows that ultrafiltration substrate concentration and ACE inhibitory activity relation curve;
Figure 8 shows that the impact that gavage ultrafiltration thing changes SHR rat blood pressure;
Figure 9 shows that the impact that gavage ultrafiltration thing changes wistar rat blood pressure;
Figure 10 shows that aminoacid sequence to be ace inhibitory peptide and the aminoacid sequence of YQKFPQYLQY be that the milk powder prepared after the ace inhibitory peptide of YS mixes by 1:1 is on the impact of SHR blood pressure;
Figure 11 shows that adding aminoacid sequence is that the ace inhibitory peptide milk powder of YQKFPQYLQY is on the impact of SHR blood pressure;
Figure 12 shows that adding aminoacid sequence is that the ace inhibitory peptide milk powder of YS is on the impact of SHR blood pressure;
Figure 13 shows that aminoacid sequence to be ace inhibitory peptide and the aminoacid sequence of YQKFPQYLQY be that the Yoghourt prepared after the ace inhibitory peptide of YS mixes by 1:1 is on the impact of SHR blood pressure;
Figure 14 shows that adding aminoacid sequence is that the ace inhibitory peptide fermented-milk of YQKFPQYLQY is on the impact of SHR blood pressure;
Figure 15 shows that adding aminoacid sequence is that the fermented-milk of the ace inhibitory peptide of YS is on the impact of SHR blood pressure.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
In the reaction process of following examples, regulated the pH value of reaction system by the HCl solution or sodium hydroxide solution dripping 1mol/L.
Embodiment 1
1. be in the caseic aqueous solution of 7% at concentration of substrate, the ratio being 1:100 according to the ratio of enzyme-to-substrate adds stomach en-, and pepsic hydrolysising condition is: temperature is 37 DEG C, pH value is 3.0, after enzymolysis 3h, terminate reaction with boiling water bath heating 10min, be cooled to room temperature.
2. the pH value to 7.7 of set-up procedure 1 gained hydrolyzed solution, then continue hydrolysis with trypsinase, the condition of tryptic hydrolysis is: temperature is 48 DEG C, and pH value is 7.7, and the ratio of Trypsin enzyme-to-substrate is 1:500; After hydrolysis 3h, hydrolyzed solution boils 10min with boiling water bath and to go out enzyme, is then cooled to 50 DEG C.The pH value reconciling hydrolyzed solution is 6.8, and add flavor protease (Novo company of Denmark) debitterize, condition is: when enzyme-to-substrate ratio is 3200U/g, hydrolysis temperature 50 DEG C, pH 6.8, hydrolysis time 1.5 are little.Hydrolyzed solution is cooled to room temperature, centrifugal 15min under 4000r/min, supernatant liquor molecular weight cut-off is the tubular fibre membrane ultrafiltration of 6Ku, and collection appears thing and carries out vacuum lyophilization, obtains the hydrolysis ultrafiltration thing that moisture is less than 5%.Namely the casein hydrolysate of ace inhibitory peptide is rich in.
3. utilize HHL (Hip-His-Leu) method vitro detection step 2 obtain the ACE inhibiting rate of product, the results are shown in Figure 7.By Fig. 7 institute fit equation y=-0.4858x
2+ 1.1035x+0.2582, wherein, R
2=0.9696, the IC of hydrolysis ultrafiltration thing can be calculated
50value is 250 μ g/mL.
4. by step 2 obtain product gavage original hypertensive rat (SHR), detect its impact on SHR blood pressure.Select male SHR rat 32 in 12 week age, weight is (260 ± 15) g, and free choice feeding is drunk water, and keeps envrionment temperature (23 ± 1) DEG C, relative humidity 60% ± 5%, raises 1 week in advance.Rat is divided into 4 groups at random, often organizes 8, be respectively blank group, low dose group, middle dosage group and high dose group, respectively gavage physiological saline (10mL/kgm
b), the hydrolysate of basic, normal, high dosage (given low is 10,50,100mg/kgm
b).Separately get Wistar rat 8, be divided into 2 groups, respectively as blank group and Normal group, gavage physiological saline and high dosage hydrolysate.Adopt BP-2006A type intelligence non-invasive blood pressure measuring (the soft grand science and technology limited Company in Beijing), measure rat tail artery systolic pressure (SBP).Before mensuration, be fixed on by rat 39 DEG C of constant temperature preheatings in mouse net muff, caudal artery is expanded, and blood flow is unimpeded.After mensuration gavage, the caudal artery blood pressure of 1 ~ 8h, surveys 3 times continuously, averages.It sees Fig. 8, Fig. 9 to the impact of blood pressure.
As can be seen from Fig. 7, Fig. 8, Fig. 9, obtain hydrolysis ultrafiltration thing, by in body and experiment in vitro, all there is hypotensive activity (p < 0.05), and on normal arterial pressure without impact (p > 0.05).
Embodiment 2
(1) at concentration of substrate be 7% caseic aqueous solution in, the ratio being 1:100 according to the ratio of enzyme-to-substrate adds stomach en-, pepsic hydrolysising condition is: temperature is 37 DEG C, pH value is 3.0, after enzymolysis 3h, terminates reaction with boiling water bath heating 10min.Then, hydrolyzed solution is cooled to 48 DEG C.Adjust pH to 7.7, then continue hydrolysis with trypsinase.The condition of tryptic hydrolysis is: temperature is 48 DEG C, and pH value is 7.7, and the ratio of Trypsin enzyme-to-substrate is 1:500; After hydrolysis 3h, hydrolyzed solution boils 10min with boiling water bath and to go out enzyme, is then cooled to room temperature.The pH value of hydrolyzed solution is regulated to be back to 7.0, centrifugal under 4000r/min, get supernatant liquor.Supernatant liquor molecular weight cut-off is the ultrafiltration membrane treatment of 6Ku, collects ultrafiltration and appears thing.Ultrafiltration is appeared thing and is separated by Sephadex G-15, obtains three components, and the active half inhibiting rate of ACE detecting three components is respectively respectively: the IC of component 1
50value is 123.41 μ g/mL; The IC of component 2
50value is 66.67 μ g/mL; The IC of component 3
50value is 64.29 μ g/mL.
(2) component 2 that selection ACE inhibitory activity is high and component 3, with LTQXL type chromatographic mass spectrometry detecting instrument (Thermo Scient ific), carry out the mass spectrometric detection of aminoacid sequence, obtain 8 fragments, with cow's milk α
1-casein, α
2-casein, beta-casein and κ-caseic amino acid alignment, is respectively N end: α
1f (25-35): FVAPFPEVFGK, α
1f (103-111): LGYLEQLLR, α
1f (178-190) YVPLGTQYTDAPSF; α
2f (56-57): YS, α
2f (98-107): YQKFPQYLQY; β f (90-102): PVVVPPFLQPEVM; κ-f (34-49): SRYPSYGLNYYQQKPV, κ-f (52-61): INNQFLPYPY.Detect the ACE inhibitory activity of each fragment respectively, wherein two fragments have ACE inhibitory activity, are respectively α
2f (56-57): YS, α
2f (98-107): YQKFPQYLQY, its ACE active half restraint IC50 value is respectively 11.89 μ g/mL and 11.75 μ g/mL.
(3) to obtained α
2f (56-57): YS fragment carries out solid phase synthesis, adopts conventional solid phase synthesis process to synthesize:
1. 2.0g Wang resin is taken in the reaction tubes of clean dried, add appropriate dimethyl formamide (DMF), activation about 30min, then take C and hold first amino acid/11 mmol, dimethyl aminopyridine (DMAP) 150mg, N, N-DIC (DIC 1) joins in reaction tubes, and DMF is solvent reaction 3h.React complete DMF to wash 4-6 time, add the mixture of appropriate pyridine and diacetyl oxide, the volume ratio of pyridine and diacetyl oxide is 1:1, reaction 30min.React complete DMF to wash 4-6 time.Then use the Fmoc of piperidine solution desamidizate acid, be total to 15min (10min+5min) de-twice.Wash 4 times with DMF again, methanol wash column 2 times, take out a small amount of resin triketohydrindene hydrate detection reagent and detect, be detected as blueness, next step reaction can be carried out.
2. take C and hold second amino acid 3mmol, tetramethyl-urea phosphofluoric acid ester (HBTU) 3mmol, in reaction tubes, adds diisopropylethylamine (DIEA) 0.5mL, reaction 40min, washes 4-6 time with DMF, and the resin triketohydrindene hydrate detection reagent that takes a morsel detects, aobvious colourless, then add piperidine solution and take off Fmoc, 10min+5min, then wash 4 times with DMF, methanol wash column twice, take out a small amount of resin triketohydrindene hydrate detection reagent to detect, be detected as blueness, next step reaction can be carried out.
3. remaining amino acid reaction method is same 2..
4. finally with trifluoroacetic acid cutting liquid cutting 2h, reaction solution suction filtration, obtains the trifluoroacetic acid solution of polypeptide, with ether sedimentation, centrifugal, get precipitation and then wash 3-5 time with ether, obtaining white solid and be the ace inhibitory peptide that aminoacid sequence is YS, through HPLC desalination, freeze-drying, the MS that takes a morsel analyzes.Obtain product purity and be greater than 98%.
(4) method identical with step (3) is adopted to carry out α
2f (98-107): the solid phase synthesis of YQKFPQYLQY, obtains the ace inhibitory peptide that aminoacid sequence is YQKFPQYLQY.
Utilize HHL (Hip-His-Leu) method vitro detection step (3) and step (4) obtain the ACE inhibiting rate of fragment, the results are shown in Figure 1 and Fig. 2.
Fig. 1 is fragment α
2the ACE inhibition of f (98-107): YQKFPQYLQY.By fit equation y=-0.0002x
2+ 0.0217x+0.2737, wherein, R
2=0.9754, ACE active half inhibiting rate IC50=11.75 μ g/mL can be calculated.Fig. 2 is fragment α
2the ACE inhibition of f (56-57): YS.By fit equation y=-9E-0.5x
2+ 0.0139x+0.2795, wherein, R
2=0.9635, ACE active half inhibiting rate IC50=11.89 μ g/mL can be calculated.
By step (3) and step (4) obtain the fragment of peptide, gavage original hypertensive rat (SHR) detects its impact on SHR blood pressure respectively.Select male SHR rat 32 in 12 week age, weight is (260 ± 15) g, and free choice feeding is drunk water, and keeps envrionment temperature (23 ± 1) DEG C, relative humidity 60% ± 5%, raises 1 week in advance.Rat is divided into 4 groups at random, often organizes 8, be respectively blank group, low dose group, middle dosage group and high dose group, respectively gavage physiological saline (10mL/kgm
b), the synthetic product of basic, normal, high dosage (given low is 2,10,20mg/kg m
b), separately get Wistar rat 8, be divided into 2 groups, respectively as blank group and Normal group, the synthetic product of gavage physiological saline and high dosage.Adopt BP-2006A type intelligence non-invasive blood pressure measuring (the soft grand science and technology limited Company in Beijing), measure rat tail artery systolic pressure (SBP).Before mensuration, be fixed on by rat 39 DEG C of constant temperature preheatings in mouse net muff, caudal artery is expanded, and blood flow is unimpeded.After mensuration gavage, the caudal artery blood pressure of 1 ~ 8h, surveys 3 times continuously, averages.It sees Fig. 3, Fig. 4, Fig. 5, Fig. 6 to the impact of blood pressure.
As can be seen from Fig. 3 and Fig. 4, obtain fragment and have original hypertensive rat (SHR) there is good hypotensive activity (p < 0.05) very well.Have Fig. 5 and Fig. 6 known, obtain fragment normal rat (wistar rat) blood pressure do not affected (p > 0.05).
Therefore, no matter external or experiment in vivo, obtain fragment and all have good hypotensive activity.
Embodiment 3
1. be for the ace inhibitory peptide of YS and aminoacid sequence are that the ace inhibitory peptide of YQKFPQYLQY is according to after the mixing of 1:1 ratio by the aminoacid sequence that embodiment 2 obtains, mix with milk powder matrix again, 0.6 gram of ace inhibitory peptide is added in every 100 grams of milk powder matrix, fully mix, obtain the milk powder with antihypertensive function.
2. by step 1 obtain milk powder, gavage original hypertensive rat (SHR), detect its impact on SHR blood pressure.Select male SHR rat 24 in 12 week age, weight is (260 ± 15) g, and free choice feeding is drunk water, and keeps envrionment temperature (23 ± 1) DEG C, relative humidity 60% ± 5%, raises 1 week in advance.Rat is divided into 3 groups at random, often organizes 8, is respectively blank group, milk powder matrix group and hypertension milk powder group, respectively gavage physiological saline (10mL/kgm
b) and 0.35g/kg m
bhypertension milk powder and milk powder matrix.Detection method is with embodiment 1.It sees Figure 10 to the impact of blood pressure.As seen from Figure 10, when with 0.35g/kg m
bthe milk powder gavage original hypertensive rat of dosage, has good antihypertensive function (p < 0.05).Within after it acts on gavage 4 hours, reach best effect (26mmHg).
Embodiment 4
1. be add the casein hydrolysate that 12.6 grams are rich in ace inhibitory peptide in 100 grams of milk powder matrix by the casein hydrolysate being rich in ace inhibitory peptide obtained in embodiment 1 and milk powder matrix with ratio, be fully the milk powder with antihypertensive function after mixing.
2. milk powder gavage original hypertensive rat step 1 obtained, detects its impact on hypertensive rat blood pressure.Detection method is with embodiment 3, and given low is 0.35g/kg m
b, the results are shown in Figure 10.As seen from Figure 10, compared with milk powder matrix, add the milk powder being rich in the casein hydrolysate of ace inhibitory peptide and there is good antihypertensive function (p < 0.05), within 4 hours after gavage, reach best effect (23mmHg).
Embodiment 5
1. be the ace inhibitory peptide of YQKFPQYLQY by the aminoacid sequence that embodiment 2 obtains, mix with milk powder matrix, add 0.6 gram of ace inhibitory peptide in every 100 grams of milk powder matrix, fully mix, obtain the milk powder with antihypertensive function.
2. by step 1 obtain milk powder, gavage original hypertensive rat (SHR), detect its impact on SHR blood pressure.Select male SHR rat 24 in 12 week age, weight is (260 ± 15) g, and free choice feeding is drunk water, and keeps envrionment temperature (23 ± 1) DEG C, relative humidity 60% ± 5%, raises 1 week in advance.Rat is divided into 3 groups at random, often organizes 8, is respectively blank group, milk powder matrix group and hypertension milk powder group, respectively gavage physiological saline (10mL/kgm
b) and 0.35g/kg m
bhypertension milk powder and milk powder matrix.Detection method is with embodiment 1.It sees Figure 11 to the impact of blood pressure.As seen from Figure 11, when with 0.35g/kg m
bthe milk powder gavage original hypertensive rat of dosage, has good antihypertensive function (p < 0.05).Within after it acts on gavage 4 hours, reach best effect (25mmHg).
Embodiment 6
1. by the ace inhibitory peptide that the aminoacid sequence that embodiment 2 obtains is for YS, mix with milk powder matrix, add 0.6 gram of ace inhibitory peptide in every 100 grams of milk powder matrix, fully mix, obtain the milk powder with antihypertensive function.
2. by step 1 obtain milk powder, gavage original hypertensive rat (SHR), detect its impact on SHR blood pressure.Select male SHR rat 24 in 12 week age, weight is (260 ± 15) g, and free choice feeding is drunk water, and keeps envrionment temperature (23 ± 1) DEG C, relative humidity 60% ± 5%, raises 1 week in advance.Rat is divided into 3 groups at random, often organizes 8, is respectively blank group, milk powder matrix group and hypertension milk powder group, respectively gavage physiological saline (10mL/kgm
b) and 0.35g/kg m
bhypertension milk powder and milk powder matrix.Detection method is with embodiment 1.It sees Figure 12 to the impact of blood pressure.As seen from Figure 12, when with 0.35g/kg m
bthe milk powder gavage original hypertensive rat of dosage, has good antihypertensive function (p < 0.05).Within after it acts on gavage 4 hours, reach best effect (27.5mmHg).
Embodiment 7
1. be for the ace inhibitory peptide of YS and aminoacid sequence are that the ace inhibitory peptide of YQKFPQYLQY is with after the mixing of 1:1 ratio by the aminoacid sequence obtained in embodiment 2, the ratio mixing of the ace inhibitory peptide of 60 milligrams is added according to the fresh milk of 100 milliliters of sterilizations, fully mix, lactic bacteria activity (YF-L812 of Hansen Corp. of 50U) is delivered directly in inoculation 0.06%, 42 DEG C ferment 5 hours, obtain the Yoghourt with antihypertensive function.
2. by step 1 obtain Yoghourt gavage original hypertensive rat (SHR), detect its impact on SHR blood pressure.Select male SHR rat 24 in 12 week age, weight is (260 ± 15) g, and free choice feeding is drunk water, and keeps envrionment temperature (23 ± 1) DEG C, relative humidity 60% ± 5%, raises 1 week in advance.Rat is divided into 3 groups at random, often organizes 8, is respectively blank group, common fermentation Yoghourt group and hypertension Yoghourt group (adding ace inhibitory peptide group), respectively gavage physiological saline (10mL/kgm
b) and 3.5g/kg m
bhypertension Yoghourt and common fermentation Yoghourt.Detection method is with embodiment 1.The results are shown in Figure 13.
As shown in Figure 13, to add and fresh milk institute after fermentation of not adding ace inhibitory peptide obtains Yoghourt, test through gavage, SHR blood pressure is affected there is significant difference (p < 0.05).The fresh milk institute after fermentation adding ace inhibitory peptide obtains Yoghourt gavage and reaches best effect (step-down 27mmHg) after 4 hours, illustrates that the Yoghourt of interpolation ace inhibitory peptide has good antihypertensive function.
Embodiment 8
1. casein hydrolysate embodiment 1 gained being rich in ace inhibitory peptide adds the ratio of 1.26 grams according to the fresh milk of 100 milliliters of sterilizations, fully mix, milk-acid bacteria (YF-L812 of Hansen Corp. of 50U) is delivered directly in inoculation 0.06%, 42 DEG C ferment 5 hours, obtain the Yoghourt with antihypertensive function.
2. by step 1 obtain Yoghourt gavage original hypertensive rat (SHR), detect its impact on SHR blood pressure.Select male SHR rat 24 in 12 week age, weight is (260 ± 15) g, and free choice feeding is drunk water, and keeps envrionment temperature (23 ± 1) DEG C, relative humidity 60% ± 5%, raises 1 week in advance.Rat is divided into 3 groups at random, often organizes 8, is respectively blank group, common fermentation Yoghourt group and hypertension Yoghourt group (the hydrolysis ultrafiltration thing of ace inhibitory peptide is rich in interpolation), respectively gavage physiological saline (10mL/kgm
b) and 3.5g/kg m
bhypertension Yoghourt and common fermentation Yoghourt.Detection method is with embodiment 1.The results are shown in Figure 13.
As shown in Figure 13, compared with common fermentation Yoghourt, add fresh milk institute after fermentation of being rich in ace inhibitory peptide casein hydrolysate and obtains Yoghourt, test through gavage, SHR blood pressure is affected there is significant difference (p < 0.05).Gavage reaches best effect (step-down 28mmHg) after 4 hours, illustrate that adding the Yoghourt being rich in ace inhibitory peptide casein hydrolysate has good antihypertensive function.
Embodiment 9
1. be the ace inhibitory peptide of YQKFPQYLQY by the aminoacid sequence obtained in embodiment 2, the ratio mixing of the ace inhibitory peptide (YQKFPQYLQY) of 60 milligrams is added according to the fresh milk of 100 milliliters of sterilizations, fully mix, lactic bacteria activity (YF-L812 of Hansen Corp. of 50U) is delivered directly in inoculation 0.06%, 42 DEG C ferment 5 hours, obtain the Yoghourt with antihypertensive function.
2. by step 1 obtain Yoghourt gavage original hypertensive rat (SHR), detect its impact on SHR blood pressure.Select male SHR rat 24 in 12 week age, weight is (260 ± 15) g, and free choice feeding is drunk water, and keeps envrionment temperature (23 ± 1) DEG C, relative humidity 60% ± 5%, raises 1 week in advance.Rat is divided into 3 groups at random, often organizes 8, is respectively blank group, common fermentation Yoghourt group and hypertension Yoghourt group (adding the fermented-milk of YQKFPQYLQY), respectively gavage physiological saline (10mL/kgm
b) and 3.5g/kg m
bhypertension Yoghourt and common fermentation Yoghourt.Detection method is with embodiment 1.The results are shown in Figure 14.
As shown in Figure 14, to add and fresh milk institute after fermentation of not adding ace inhibitory peptide obtains Yoghourt, test through gavage, SHR blood pressure is affected there is significant difference (p < 0.05).The fresh milk institute after fermentation adding ace inhibitory peptide obtains Yoghourt gavage and reaches best effect (step-down 31.37mmHg) after 4 hours, illustrates that the Yoghourt of interpolation ace inhibitory peptide has good antihypertensive function.
Embodiment 10
1. be the ace inhibitory peptide of YS by the aminoacid sequence obtained in embodiment 2, the ratio mixing of the ace inhibitory peptide (YS) of 60 milligrams is added according to the fresh milk of 100 milliliters of sterilizations, fully mix, lactic bacteria activity (YF-L812 of Hansen Corp. of 50U) is delivered directly in inoculation 0.06%, 42 DEG C ferment 5 hours, obtain the Yoghourt with antihypertensive function.
2. by step 1 obtain Yoghourt gavage original hypertensive rat (SHR), detect its impact on SHR blood pressure.Select male SHR rat 24 in 12 week age, weight is (260 ± 15) g, and free choice feeding is drunk water, and keeps envrionment temperature (23 ± 1) DEG C, relative humidity 60% ± 5%, raises 1 week in advance.Rat is divided into 3 groups at random, often organizes 8, is respectively blank group, common fermentation Yoghourt group and hypertension Yoghourt group (adding the fermented-milk of YS), respectively gavage physiological saline (10mL/kgm
b) and 3.5g/kg m
bhypertension Yoghourt and common fermentation Yoghourt.Detection method is with embodiment 1.The results are shown in Figure 15.
As shown in Figure 15, to add and fresh milk institute after fermentation of not adding ace inhibitory peptide obtains Yoghourt, test through gavage, SHR blood pressure is affected there is significant difference (p < 0.05).The fresh milk institute after fermentation adding ace inhibitory peptide obtains Yoghourt gavage and reaches best effect (step-down 32.5mmHg) after 4 hours, illustrates that the Yoghourt of interpolation ace inhibitory peptide has good antihypertensive function.
The casein hydrolysate being rich in ace inhibitory peptide of gained of the present invention and ace inhibitory peptide have the significantly hypertensive effect of reduction, gained is rich in the casein hydrolysate of ace inhibitory peptide and ace inhibitory peptide to add in milk-product there is remarkable antihypertensive effect, on normal arterial pressure without impact, edible safety, is applicable to Hypertensive Population and eats.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (7)
1. an ace inhibitory peptide, its aminoacid sequence is: YS.
2. a preparation method for ace inhibitory peptide according to claim 1, is characterized in that, comprises the steps:
(1) at concentration of substrate be 5-9% caseic aqueous solution in, the ratio being 1:100 according to the ratio of enzyme-to-substrate adds stomach en-, pepsic hydrolysising condition is: temperature is 37 DEG C, pH value is 2.0-3.0, after hydrolysis 3h, terminate reaction with boiling water bath heating 10min, be cooled to 45-50 DEG C;
(2) pH value of set-up procedure (1) gained hydrolyzed solution is to 7.7-8.5, then continues hydrolysis with trypsinase, and tryptic hydrolysising condition is: temperature is 48 DEG C, and pH value is 7.7-8.5, and the ratio of Trypsin enzyme-to-substrate is 1:500; After hydrolysis 3h, hydrolyzed solution boils 10min with boiling water bath and to go out enzyme, is then cooled to room temperature; The pH value of regulation system is back to 7.0; Afterwards, centrifugal, get supernatant liquor;
(3) step (2) gained supernatant liquor is carried out uf processing, molecular weight cut-off is 6Ku, collects ultrafiltration and appears thing;
(4) ultrafiltration is appeared thing and is separated by Sephadex G-15, obtains three components, detects the ACE inhibitory activity of three components respectively;
(5) select the high component chromatographic mass spectrometry detecting instrument of the middle ACE inhibitory activity of step (4) to carry out the mass spectrometric detection of aminoacid sequence, obtaining aminoacid sequence is the fragment of YS; Adopt solid phase synthesis technique to synthesize, obtain the bioactive peptide with ACE inhibit feature.
3. be rich in an antihypertensive milk powder for ace inhibitory peptide, it is characterized in that, in milk powder matrix, add the ace inhibitory peptide that aminoacid sequence is YS.
4. the antihypertensive milk powder being rich in ace inhibitory peptide according to claim 3, is characterized in that, adding 0.6 gram of aminoacid sequence in 100 grams of milk powder matrix is the ace inhibitory peptide of YS.
5. be rich in an antihypertensive Yoghourt for ace inhibitory peptide, it is characterized in that, in fresh milk, add the ace inhibitory peptide that aminoacid sequence is YS.
6. the antihypertensive Yoghourt being rich in ace inhibitory peptide according to claim 5, is characterized in that, adding 60 milligrams of aminoacid sequences in 100 ml fresh milks is the ace inhibitory peptide of YS.
7. a preparation method for antihypertensive Yoghourt according to claim 5, is characterized in that, comprise the steps:
(1) in the fresh milk through sterilization, add the ace inhibitory peptide that aminoacid sequence is YS, fully mix;
(2) inoculate 0.06% and deliver directly milk-acid bacteria, 42 DEG C ferment 5 hours, obtain the Yoghourt with antihypertensive function.
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Cited By (5)
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| CN107163130A (en) * | 2017-06-08 | 2017-09-15 | 广西大学 | A kind of inhibiting peptide of tonin and its preparation extracting method |
| CN107173438A (en) * | 2017-06-09 | 2017-09-19 | 天津商业大学 | A kind of production method with aided blood pressure-lowering and liver-protecting function Yoghourt |
| CN110386960A (en) * | 2019-07-29 | 2019-10-29 | 内蒙古塞飞亚农业科技发展股份有限公司 | Duck protein sources have the p277 SPAF and purposes of ACE and HMG-CoA reductase inhibitory activity |
| CN110396123A (en) * | 2019-07-29 | 2019-11-01 | 内蒙古塞飞亚农业科技发展股份有限公司 | Duck protein sources have ACE inhibitory activity p277 SYVP and purposes |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107163130A (en) * | 2017-06-08 | 2017-09-15 | 广西大学 | A kind of inhibiting peptide of tonin and its preparation extracting method |
| CN107173438A (en) * | 2017-06-09 | 2017-09-19 | 天津商业大学 | A kind of production method with aided blood pressure-lowering and liver-protecting function Yoghourt |
| CN110386960A (en) * | 2019-07-29 | 2019-10-29 | 内蒙古塞飞亚农业科技发展股份有限公司 | Duck protein sources have the p277 SPAF and purposes of ACE and HMG-CoA reductase inhibitory activity |
| CN110396123A (en) * | 2019-07-29 | 2019-11-01 | 内蒙古塞飞亚农业科技发展股份有限公司 | Duck protein sources have ACE inhibitory activity p277 SYVP and purposes |
| CN114671939A (en) * | 2022-04-02 | 2022-06-28 | 华南理工大学 | ACE inhibitory peptide with mild, stable and long-acting antihypertensive effect and application thereof |
| CN114671939B (en) * | 2022-04-02 | 2023-09-26 | 华南理工大学 | ACE (angiotensin converting enzyme) inhibitory peptide with mild blood pressure lowering effect, stable effect and long acting and application thereof |
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