CN109207538A - The preparation method and applications of pea protein Antihypertensive Peptides - Google Patents
The preparation method and applications of pea protein Antihypertensive Peptides Download PDFInfo
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- CN109207538A CN109207538A CN201810900244.3A CN201810900244A CN109207538A CN 109207538 A CN109207538 A CN 109207538A CN 201810900244 A CN201810900244 A CN 201810900244A CN 109207538 A CN109207538 A CN 109207538A
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Abstract
The invention belongs to technical field of nutritional food, and in particular to a kind of preparation method and applications of pea protein Antihypertensive Peptides.The present invention shortens enzymolysis time, substantially increases enzymolysis efficiency by carrying out squeezing crushing to Fed Protein Powder of Pea Insteal and preparing pea protein Antihypertensive Peptides using neutral proteinase progress enzyme digestion reaction.The pea protein Antihypertensive Peptides can effectively inhibit the activity of Angiotensin-Converting (ACE), so can be used as Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe to reduce blood pressure.Pea protein Antihypertensive Peptides of the invention not only have the function of efficient antihypertensive effect, nontoxicity, without side-effects etc., also have many advantages, such as to adjust human metabolism, immune function, anti-aging, and only have effect to hypertensive patient, it has no adverse effects to normotensive.The pea protein Antihypertensive Peptides later period of the invention can prepare functional food, replace conventional medicament, avoid traditional chemical side effects of pharmaceutical drugs to human body bring adverse effect.
Description
Technical field
The invention belongs to technical field of nutritional food, and in particular to pea protein Antihypertensive Peptides preparation method and applications.
Technical background
Pea protein is a kind of good protein, and amino acid ratio relatively balances, and in addition to methionine content is slightly lower, remaining must
Amino acid is needed to reach FAO/WHO recommendation pattern value " 1 ".In addition, pea protein has preferable water-retaining property, oil absorption and gel shape
It becomes second nature, can be applied in various functional foods.But so far.China is that extraction and application pea forms sediment to pea processing emphasis
Powder, preparing the albumen after starch in residue not yet can be carried out abundant development and utilization, at present overwhelming majority residues only make feed it
With causing valuable protein resource waste, so preparing Antihypertensive Peptides from pea protein, resource can be made full use of, become useless
For precious, reduction waste.
Count according to investigations, population of China Prevalence of Hypertension is in obvious ascendant trend over 50 years, China in 2016 18 years old and
The above adult hypertension illness rate is 25.2%, every year newly-increased number of patients 10,000,000 or so.China hypertensive patient number at present
More than 300,000,000.Based on the status of China's Prevalence of Hypertension, the treatment of hypertension is a problem of crucial importance.
Angiotensin-Converting (Angiotensin I-converting Enzyme, abbreviation ACE) is to adjusting blood pressure
Critical effect is played, angiotensin I (AngiotensinI, abbreviation AI) does not have activity, but AI is in the effect of ACE
It can be changed into down active Angiotensin II (AngiotensinII, abbreviation AII), AII has vasoconstriction smooth
The active function of flesh, so as to cause blood pressure raising.Due to internal ACE activity it is too high caused by hypertension, inhibition can be passed through
ACE activity prevents the reduction of generation and the kassinin kinin degradation of Angiotensin II, to have the function that treat hypertension.Therefore,
Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe can be used as the drug of blood pressure lowering.
It is reported that many developed countries attempt the inhibitor of chemical synthesis ACE already, and use in clinical treatment.So
And chemically synthesized antihypertensive drugs often causes the various secondary works such as fash, throat swelling and pain, gustatory system imbalance, kidney damage
With, be not suitable for continuously take for a long time.Research shows that, it is already possible to divide from animal protein, vegetable protein, microbial metabolic products
From extraction blood pressure lowering peptide.Food-borne picture extracts Antihypertensive Peptides compared with chemically synthesized blood-pressure drug from pea protein, has nothing
The advantages that side effect, good absorbing simultaneously only have antihypertensive effect to pathologic hypertensive patient, without side-effects to normal body.
There are some researches prove people to have prepared efficient ace inhibitory peptide from the raw material of separate sources respectively, to plant egg
The research of Bai Yuan has soybean, corn, peanut, rice bran etc., and but there are no about the research for preparing Antihypertensive Peptides from pea protein.
Chinese hypertension incidence crowd increases year by year, and hypertension medication market can further increase.Currently, hypertensive patient
In know and carry out pharmaceutical intervention number accounting it is less, it is high with the continuous promotion that medical level and people's medical treatment are realized
Blood pressure medication market can further open, and have a extensive future.
Summary of the invention
The object of the present invention is to provide a kind of pea protein Antihypertensive Peptides preparation method and applications, by Fed Protein Powder of Pea Insteal
It carries out squeezing crushing and carries out enzyme digestion reaction using neutral proteinase to prepare Antihypertensive Peptides, shorten enzymolysis time, improve enzyme
Solution efficiency, the DPPH clearance rate of pea peptide,.OH clearance rate and O2 .-Clearance rate is all greatly improved；And it is studied to height
The inhibiting effect of blood pressure only has antihypertensive effect to hypertensive patient, and does not influence on normotensive body, highly-safe
And it has no toxic side effect.
The preparation method of pea protein Antihypertensive Peptides of the present invention: extrusion process are as follows: the pea for being 28% ~ 30% by water content
Legumin powder is squeezed under conditions of temperature is 53 ~ 55 DEG C, screw speed is 170 ~ 175 r/min, fenestra aperture is 10 mm
Pressure is crushed to 50 mesh after Fed Protein Powder of Pea Insteal after extruding is cooling.
Enzymolysis process are as follows: smashed Fed Protein Powder of Pea Insteal will be squeezed and be dissolved in the phosphate buffer solution that pH value is 6.7 ~ 7
In, then with neutral proteinase it is digested, obtain enzymolysis product.
Wherein: neutral protease enzymolysis optimum condition: enzymolysis temperature is 40 ~ 42 DEG C, and the pH value of enzyme digestion reaction is 6.7
~ 7, enzyme concentration is 12.5% ~ 13.2 %(E/S), pea protein concentration is 6.8% ~ 6.92 %(g/mL in enzyme digestion reaction), when enzymatic hydrolysis
Between be 2.6 ~ 3 h.
After enzymatic hydrolysis, enzyme-removal temperature is 100 DEG C, and the enzyme deactivation time is 10 min.
Pea protein Antihypertensive Peptides are separated using bag filter: using the bag filter of 7 KDa, 3.5 KDa, 1 KDa, to enzymolysis liquid
Successively separate.Bag filter is pre-processed, about 2/3rds enzymolysis liquid, two end seals are added into the bag filter handled well
It is good, it is placed in distilled water and is stirred, change a water every 0.5h, there is no color changes up to peritoneal effluent, and peritoneal effluent is used
After Rotary Evaporators concentration, concentrate is freeze-dried, and obtains pea protein decompression Gly-His-Lys.
Wherein, enzymolysis product obtains four fractions through dialysis: isolated less than 1 KDa, 1~3.5 KDa, 3.5~7
KDa and enzymolysis liquid greater than 74 different components of KDa.Measure its O2 .-Free radical scavenging activity is respectively 83%, 35%, 21%,
14%.It was found that molecular weight is most strong less than the enzymolysis liquid activity of 1 KDa, molecular weight is had focused largely in 200~800 Da or so, is said
The peptide obtained after bright enzymatic hydrolysis is 2~8 peptides mostly.
After enzyme digestion reaction, the DPPH clearance rate of pea peptide,.OH clearance rate, O2 .-Clearance rate is respectively 98%, 83%, 76%.
The application of pea protein Antihypertensive Peptides of the present invention: the additive amount of 2-3 mg pea protein Antihypertensive Peptides is to 1 unit
The inhibiting rate highest of ACE enzyme activity, inhibiting rate is 37% ~ 39% at this time.
There is obvious antihypertensive effect to hypertensive patient, can add and antihypertensive drugs or the nutrition food with buck functionality is made
Product.
Compared with prior art, beneficial effects of the present invention are as follows:
(1) in the present invention, pea protein is crushed by squeezing, and certain variation has occurred in space structure, and chain structure ratio increases,
Hole increases, and the concave-convex sense on surface is more strong, and specific area increases, so the contact area of pea protein and neutral proteinase is more
Greatly, be conducive to enzyme to enter inside pea protein, reduce enzymolysis time, improve enzyme digestion reaction efficiency.
(2) in the present invention, the Antihypertensive Peptides extracted from pea protein only suffer from hypertension as a kind of biologically active polypeptide
Person has antihypertensive effect, and does not influence on normotensive body, highly-safe and have no toxic side effect.Traditional change can be replaced
Learn antihypertensive drugs.
(3) in the present invention, pea protein peptide, which also has, improves the function such as human metabolism, strengthen immunity, anti-aging
Can, it is useful and harmless to human body.
Specific embodiment
The present invention is described further with reference to embodiments.
Embodiment 1
The preparation method of pea protein Antihypertensive Peptides: in temperature being 55 DEG C, screw speed by the Fed Protein Powder of Pea Insteal that water content is 30%
It is squeezed under conditions of being 10 mm for 175 r/min, fenestra aperture, Fed Protein Powder of Pea Insteal after extruding is crushed to 50 through cooling
Mesh.
It weighs the pea protein extrudate sample of 3.46 g, the neutral proteinase of 6.6 g is added and is dissolved in pH value is
It in 6.99 phosphate buffer solution, shakes up, is placed in constant temperature oscillation water-bath, oscillation 3 h of enzymatic hydrolysis, after, boiling water bath goes out
10 min of enzyme.After cooling, protein enzymatic hydrolyzate is filtered, takes filtrate stand-by.
This experiment uses the bag filter of 1 KDa, is successively separated to enzymolysis liquid.Bag filter is pre-processed, to place
About 2/3rds enzymolysis liquid is added in the bag filter managed, both ends are sealed, and are placed in distilled water and are stirred, when one section
Between change a water, up to peritoneal effluent, there is no color changes, and peritoneal effluent is concentrated with Rotary Evaporators, is freezed to concentrate
Dry 24 h obtain pea protein decompression Gly-His-Lys.
Wherein:
The neutral protease enzymolysis reaction temperature for preparing pea protein Antihypertensive Peptides is 40 DEG C, and the pH value of enzyme digestion reaction is 6.7, is added
Enzyme amount is 13.2 %(E/S), and pea protein concentration is 6.92 %(g/mL in enzyme digestion reaction), enzymolysis time is 3 h.
After enzymatic hydrolysis, enzyme-removal temperature is 100 DEG C, and the enzyme deactivation time is 10 min.
PH value be 6.99 phosphate buffer solution by 0.2 M Na2HPO4With the NaH of 0.2 M2PO4It is formulated.
After enzyme digestion reaction, the DPPH clearance rate of pea peptide,.OH clearance rate, O2 .-Clearance rate is respectively 95%, 88%, 45%.
Measurement of the pea protein Antihypertensive Peptides to ACE enzyme inhibition rate: using the measuring method of Cushman, in the centrifugation of 2 mL
Guan Zhong, the total volume tested every time are 0.2 mL.HHL is dissolved in borate of 0.1 moL/L containing 0.3 moL/L sodium chloride to delay
In fliud flushing (pH=8.3), it is configured to hippuroyl histidyl- leucine (HHL) solution for standby that concentration is 5 mmoL/L.It is being centrifuged
The HHL of 80 μ L, 100 μ L deionized waters are sequentially added in pipe, 10 uL pea proteins are depressured peptide hydrolysate (0. 20 mg/mL),
This mixed liquor is put into 37 DEG C of waters bath with thermostatic control and keeps the temperature 3 min, the starting reaction of 10 μ L ACE enzyme solutions is then added.Constant temperature is kept
30 min, be added 0.2 mL 1 moL/L HCL terminate reaction, add 20 DEG C of 1.2 mL(-) ethyl acetate, uniformly
About 15 s are mixed, to extract the hippuric acid (Hip) discharged by ACE, finally, taking out after centrifugation (3500 r/min) 5 min
0.8 mL ester layer, is transferred in another test tube, and 1 h or so is dried in 120 °C of baking oven, be redissolved in 0.8 mL go from
In sub- water, light absorption value is measured at 228 nm.
For blank control pipe in addition to the 1 moL/L HCL that 0.2 mL is first added before the reaction terminates reaction, remaining operation is identical.
Wherein, inhibiting rate calculating formula is as follows: inhibiting rate (%)=(ODA-ODB)/(ODA-ODC)
ODBFor there are light absorption values when inhibitor and enzyme；ODAFor there is no light absorption value when inhibitor, (deionized water replaces water
Solve liquid)；ODCBy inhibitor and enzyme all in the absence of light absorption value (replace hydrolyzate with deionized water, and before the reaction plus hydrochloric acid
Terminate reaction).
Pea protein Antihypertensive Peptides are 37.1% to ACE enzyme inhibition rate.
Embodiment 2
The preparation method of pea protein Antihypertensive Peptides: in temperature being 53 DEG C, screw speed by the Fed Protein Powder of Pea Insteal that water content is 28%
It is squeezed under conditions of being 10 mm for 170 r/min, fenestra aperture, Fed Protein Powder of Pea Insteal after extruding is crushed to 50 through cooling
Mesh.
The pea protein extrudate sample of 3.46 g is weighed, it is 6.7 that the neutral proteinase of 6.6 g, which is added, and is dissolved in pH value
Phosphate buffer solution in, shake up, be placed in constant temperature oscillation water-bath, oscillation enzymatic hydrolysis 2.6 h, after, boiling water bath enzyme deactivation
10 min.After cooling, protein enzymatic hydrolyzate is filtered, takes filtrate stand-by.
This experiment uses the bag filter of 1 KDa, is successively separated to enzymolysis liquid.Bag filter is pre-processed, to place
About 2/3rds enzymolysis liquid is added in the bag filter managed, both ends are sealed, and are placed in distilled water and are stirred, when one section
Between change a water, up to peritoneal effluent, there is no color changes, and peritoneal effluent is concentrated with Rotary Evaporators, is freezed to concentrate
Dry 24 h obtain pea protein decompression Gly-His-Lys.
Wherein:
The neutral protease enzymolysis reaction temperature for preparing pea protein Antihypertensive Peptides is 42 DEG C, and the pH value of enzyme digestion reaction is 6.7, is added
Enzyme amount is 12.5 %(E/S), and pea protein concentration is 6.8%(g/mL in enzyme digestion reaction), enzymolysis time is 3 h.
After enzymatic hydrolysis, enzyme-removal temperature is 100 DEG C, and the enzyme deactivation time is 10 min.
PH value be 6.7 phosphate buffer solution by 0.2 M Na2HPO4With the NaH of 0.2 M2PO4It is formulated.
After enzyme digestion reaction, the DPPH clearance rate of pea peptide,.OH clearance rate, O2 .-Clearance rate is respectively 94%, 88%, 46%.
Measurement of the pea protein Antihypertensive Peptides to ACE enzyme inhibition rate:
Experiment carries out in the centrifuge tube of 2 mL, and it is 8.3 that the total volume tested every time, which is 0.2 mL: the pH containing 50 mmoL/L,
The pea protein of HEPES buffer solution, 300 mmoL/L NaCl, 5 mmoL/L HHL, 0. 25 mg/mL is depressured peptide hydrolysate 10
UL and 100 uL deionized waters keep the temperature 5 min in 37 DEG C of waters bath with thermostatic control, and the ACE enzyme solution of 10 μ L is then added, and (0.1 U is dissolved in
In 1 mL same buffer) starting reaction, after constant temperature keeps 30 min, the 1 moL/L HCl that 0.2 mL is added terminates reaction.Again
Ethyl acetate after 1.2 mL freezing is added, uniformly mixes 15 s, and 0.8 mL ester is taken out in centrifugation (3500 r/min, 5 min) afterwards
Layer is transferred in another test tube, is dried in 120 DEG C of baking oven through 1 h, then it is redissolved in the deionized water of 0.8 mL,
Light absorption value is measured at 228 nm.
Enzyme-activity unit definition: 1 unit enzyme activity is defined as being catalyzed HHL 1 umoL hippuric acid of formation in 37 DEG C, 1 min
Enzyme amount.
Wherein, inhibiting rate calculating formula is as follows: inhibiting rate (%)=(ODA-ODB)/(ODA-ODC)
ODBFor there are light absorption values when inhibitor and enzyme；ODAFor there is no light absorption value when inhibitor, (deionized water replaces water
Solve liquid)；ODCBy inhibitor and enzyme all in the absence of light absorption value (replace hydrolyzate with deionized water, and before the reaction plus hydrochloric acid
Terminate reaction).
Pea protein Antihypertensive Peptides are 39.2% to ACE enzyme inhibition rate.
Claims (10)
1. a kind of preparation method and applications of pea protein Antihypertensive Peptides, it is characterised in that: made by the enzymatic hydrolysis of neutral proteinase
With from pea protein extraction prepare Antihypertensive Peptides, detect its inhibiting effect to ACE, it was demonstrated that pea protein Antihypertensive Peptides of the invention
There is good antihypertensive effect to hypertensive patient.
2. a kind of preparation method and applications of pea protein Antihypertensive Peptides according to claim 1, it is characterised in that: squeeze
Process are as follows: by water content be 28% ~ 30% Fed Protein Powder of Pea Insteal temperature be 53 ~ 55 DEG C, screw speed be 170 ~ 175 r/min,
Fenestra aperture is squeezed under conditions of being 10 mm, is crushed to 50 mesh after Fed Protein Powder of Pea Insteal after extruding is cooling.
3. a kind of preparation method and applications of pea protein Antihypertensive Peptides according to claim 1, it is characterised in that: enzymatic hydrolysis
Process are as follows: will squeeze smashed Fed Protein Powder of Pea Insteal be dissolved in pH value be 6.7 ~ 7 phosphate buffer solution in, then with neutrality egg
White enzyme digests it, obtains enzymolysis product, in which: neutral protease enzymolysis optimum condition: enzymolysis temperature is 40 ~ 42
DEG C, the pH value of enzyme digestion reaction is 6.7 ~ 7, and enzyme concentration is 12.5% ~ 13.2 %(E/S), and pea protein concentration is in enzyme digestion reaction
6.8% ~ 6.92 %(g/mL), enzymolysis time is 2.6 ~ 3 h, and after enzymatic hydrolysis, enzyme-removal temperature is 100 DEG C, and the enzyme deactivation time is 10
min。
4. a kind of preparation method and applications of pea protein Antihypertensive Peptides according to claim 1, it is characterised in that: utilize
Bag filter separates pea protein Antihypertensive Peptides: using the bag filter of 7 KDa, 3.5 KDa, 1 KDa, successively separating to enzymolysis liquid.
5. bag filter is pre-processed, about 2/3rds enzymolysis liquid is added into the bag filter handled well, both ends are sealed, and are put
It is stirred in distilled water, changes a water every 0.5h, until there is no color changes for peritoneal effluent, peritoneal effluent rotation is steamed
After sending out instrument concentration, concentrate is freeze-dried, and obtains pea protein decompression Gly-His-Lys.
6. a kind of preparation method and applications of pea protein Antihypertensive Peptides according to claim 1, it is characterised in that: wherein
Collection molecular weight is most strong less than the enzymolysis liquid activity of 1 KDa, and molecular weight has focused largely in 200~800 Da or so, 2~8 peptides.
7. its DPPH clearance rate,.OH clearance rate, O2 .-Clearance rate is respectively 98%, 83%, 76%.
8. a kind of preparation method and applications of pea protein Antihypertensive Peptides according to claim 1, it is characterised in that: pea
The additive amount of protein antihypertensive peptide is that every 1 unit ACE enzyme activity adds 2 ~ 3 mg pea protein Antihypertensive Peptides.
9. a kind of preparation method and applications of pea protein Antihypertensive Peptides according to claim 1, it is characterised in that: pea
Protein antihypertensive peptide and ACE enzyme reaction temperature are 35 ~ 37.5 DEG C, and the reaction time is 30 min.
10. a kind of preparation method and applications of pea protein Antihypertensive Peptides according to claim 1, it is characterised in that: benefit
Obtain pea protein Antihypertensive Peptides with neutral protease enzymolysis, pea protein Antihypertensive Peptides to the inhibiting rate of ACE enzyme be 37.1% ~
39.2%。
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| CN114277077A (en) * | 2021-12-28 | 2022-04-05 | 黑龙江八一农垦大学 | Compound ACE inhibitory peptide with synergistic effect and preparation method and application thereof |
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| CN114277077A (en) * | 2021-12-28 | 2022-04-05 | 黑龙江八一农垦大学 | Compound ACE inhibitory peptide with synergistic effect and preparation method and application thereof |
| CN114277077B (en) * | 2021-12-28 | 2023-10-27 | 黑龙江八一农垦大学 | Composite ACE (angiotensin converting enzyme) inhibitory peptide with synergistic effect as well as preparation method and application thereof |
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Application publication date: 20190115 |