CN110734947A - preparation method of oat blood pressure lowering polypeptide - Google Patents
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Abstract
The invention belongs to the technical field of food processing, and particularly relates to a preparation method of oat blood pressure-lowering polypeptides.
Description
Technical Field
The invention belongs to the technical field of food processing, and particularly relates to a preparation method of oat blood pressure reducing polypeptides.
Background
In recent years, with the improvement of living standard of people, the incidence of hypertension is increased year by year, which has great harm to human body, can cause the change and damage of the structure and function of heart, brain, kidney, blood vessel and eye, causes various complications, becomes an important factor of cardiovascular and cerebrovascular diseases and the like, and seriously harms the life health of people.
The existing commonly used blood pressure lowering medicines mainly comprise 6 categories of α 1-receptor blockers, CCBs, ACEIs, ARBs, non-selective β -receptor blockers, diuretics and the like, although western medicines have good effects and quick effects in treating the diseases, the hypertension is taken as a long-term chronic disease, the toxic and side effects of the western medicines are obvious after long-term administration, and is harmful to human bodies, so that the development of novel safe and efficient blood pressure lowering medicines is necessary.
The invention discloses quinoa polypeptides with blood pressure lowering effect and a preparation method thereof, wherein the preparation method comprises the following steps of extracting quinoa protein from defatted quinoa powder by an alkali extraction and acid precipitation method, and performing compound enzymolysis, ultrafiltration, desalination, vacuum concentration and freeze drying to obtain the quinoa blood pressure lowering active peptide.
The invention patent CN 101117642 discloses blood pressure lowering polypeptides and a preparation method thereof, wherein the method comprises the following steps of 1) suspending oat bran in water, adjusting the pH value to 8.5-9.5, heating at 50-60 ℃, 2) adding alkaline protease into the pretreated liquid, stirring and carrying out enzymolysis for 3-5 hours at 50-60 ℃, 3) centrifuging the enzymolysis liquid obtained in the step 2, taking supernatant, adding β -glucanase, reacting medium temperature amylase and glucoamylase for 2-4 hours, filtering, adsorbing the filtrate by macroporous adsorption resin, washing with deionized water to remove sugar and salt, and eluting by using 75% by volume of ethanol solution to obtain the blood pressure lowering polypeptides.
Aiming at the defects, the invention provides a feasible way for alleviating the harm of the cardiovascular and cerebrovascular diseases in the simplest, economic and safe way.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of oat antihypertensive polypeptides, so as to overcome the defects of the existing method that western medicines are not relied on and the existing preparation method of oat antihypertensive polypeptides is not relied on, and safe, healthy and side-effect-free antihypertensive oat polypeptides and the preparation method thereof are provided.
In order to achieve the purpose, the technical scheme of the invention is as follows:
A method for preparing oat antihypertensive polypeptide, comprising the following steps:
(1) extraction of oat protein
Pulverizing oat grains, sieving, adding water to dissolve, adjusting pH to 10.0, leaching in constant temperature water bath, centrifuging, separating to obtain supernatant, adjusting pH of the supernatant to isoelectric point of protein, centrifuging to obtain protein precipitate, and drying to obtain oat protein dry powder;
(2) enzymolysis
Preparing the oat protein dry powder prepared in the step (1) into aqueous solution, placing the aqueous solution in a constant-temperature water bath kettle, uniformly mixing the aqueous solution with an electric stirrer, adjusting the pH value to 9-10, adding enzyme for enzymolysis, inactivating the enzyme, cooling, centrifuging, and taking supernatant;
(3) method for obtaining oat antihypertensive polypeptide
Separating the supernatant obtained in the step (2) by a membrane ultrafiltration method, collecting peptide fragments, and drying to obtain the powdery oat antihypertensive polypeptide.
Preferably, in the step (1), after the oat grains are crushed, the oat grains are sieved by a 60-mesh sieve, and the mixture ratio of the oat grains to the feed liquid is 1: 9, adding water for dissolving; the pH was adjusted to 10.0 using 1mol/L NaOH.
Preferably, in the step (1), the leaching condition of the thermostatic waterbath is that the leaching is carried out for 120min in a water bath kettle at 50 ℃; after being leached by constant temperature water bath, the mixture is centrifuged for 15min at 4500 r/min.
Preferably, in the step (1), the pH of the supernatant is adjusted to 4.0 by using 1mol/L HCl, so that the pH is adjusted to the isoelectric point of the protein; centrifuging at 4500r/min for 15min, and collecting protein precipitate; the drying method is vacuum freeze drying.
Preferably, in the step (2), the oat protein dry powder is prepared into an aqueous solution with the mass concentration of 8% -10%; the pH is adjusted to 9-10 with 0.1mol/L NaOH solution.
Preferably, in step (2), Alcalase 2.4L FG is selected as an alkaline protease for enzymolysis.
Preferably, in the step (2), the enzymolysis conditions are as follows: when the temperature reaches 50-60 ℃, adding 2.4L FG alkaline protease Alcalase according to the volume ratio of 1.5-2.0mL/L, and performing enzymolysis for 90-150 min; the alkaline protease Alcalase 2.4L FG has an enzyme unit of 2.4 AU.
Preferably, in the step (2), the enzyme deactivation conditions are as follows: inactivating enzyme at 100 deg.C for 15 min; after cooling, centrifuging for 10min at 10000 r/min.
Preferably, in step (3), the peptide fragment with a molecular weight of <5kDa is collected.
Preferably, in the step (3), the drying method is vacuum freeze drying; the collected powdery oat antihypertensive polypeptide is stored at the temperature of-20 ℃, and is dissolved by normal saline to be prepared into the required concentration when in use.
Compared with the prior art, the invention has the advantages that:
(1) the oat is a worldwide grain crop, the yield of the oat is the seventh place in the grain crop, China is a main country for producing the oat, the yield of the oat is high, the price is relatively low, and the oat is easy to obtain.
(2) The oat antihypertensive polypeptide contains 17 amino acids, 8 of which are essential amino acids for human bodies, and can improve the nutritional condition of people and promote the improvement of the health level of people.
(3) The oat antihypertensive polypeptide prepared by the invention contains rich food-borne ACE inhibitory peptide, has high in vitro ACE inhibitory rate, is safe, free of side effect and easy to absorb, can reduce blood pressure and blood fat, has no influence on people with normal blood pressure, and has important significance for research on oat ACE inhibitory peptide.
In conclusion, after long-term eating, the oat antihypertensive polypeptide prepared by the invention can effectively prevent the risk of hypertension and hyperlipidemia, has definite antihypertensive effect on people already suffering from hypertension, and is safe and free of toxic and side effects.
Drawings
FIG. 1 is a graph of the results of a single depressurization experiment characterized by systolic pressure;
FIG. 2 is a graph of the results of a single hypotensive experiment characterized by diastolic blood pressure;
FIG. 3 is a graph of the results of a 42-day hypotensive experiment characterized by systolic blood pressure;
FIG. 4 is a graph of the results of a 42-day hypotensive experiment characterized by diastolic blood pressure.
Detailed Description
(1) Experimental materials:
oat grains, Shijiazhuang Lingfeng agricultural and sideline products development Limited company in Hebei province;
alcalase 2.4L FG (181,194U/g), Novoxil Biotechnology Ltd; sodium hydroxide, hydrochloric acid.
(2) An experimental instrument:
PHS-3C precision pH meter, Shanghai precision scientific instruments, Inc.;
HH constant temperature water bath, Jiangsu Jinda City large and medium instrument factory;
JJ-1 precision force-increasing electric stirrer, jin Tan Ke-Shi analyzer Co., Ltd;
TG16-WS desk-top high-speed centrifuge, Changshan apparatus centrifuge instruments GmbH;
FC160 hammer mill, shanghai chinese drug machinery plant;
t16 New century ultraviolet spectrophotometer, Beijing Pujingyan general instruments, Limited liability company;
FreeZone 12plusa vacuum freeze dryer, Labconco, USA;
ALC-NIBP non-invasive blood pressure measurement and analysis system, Shanghai Oercote Biotech limited;
BioTek Eon TM microplate spectrophotometer, Biotek Instruments, Ins, USA;
tissue grinder, high throughput tissue grinder, QIAGEN, germany, tissue;
ALLEGRAX-12R Universal desktop refrigerated centrifuge, Beckman Coulter, USA.
The invention is described in further detail with reference to the figures and the detailed description.
Example 1
A method for preparing oat antihypertensive polypeptide, comprising the following steps:
(1) extraction of oat protein
Crushing oat grains, sieving the crushed oat grains with a 60-mesh sieve, and mixing the crushed oat grains with a feed-liquid ratio of 1: 9, adding water for dissolving; adjusting pH to 10.0 with 1mol/L NaOH, leaching at constant temperature in 50 deg.C water bath for 120min, centrifuging at 4500r/min for 15min, and separating supernatant (i.e. removing precipitate); adjusting the pH of the supernatant to 4.0 (protein isoelectric point) with 1mol/L HCl, centrifuging at 4500r/min for 15min, and collecting protein precipitate (i.e. removing supernatant); and (3) carrying out vacuum freeze drying on the protein precipitate to obtain oat protein dry powder.
(2) Enzymolysis
Preparing the oat protein dry powder obtained in the step (1) into an aqueous solution with the mass concentration of 10%; placing in a constant temperature water bath kettle, mixing with an electric stirrer, adjusting pH to 9.5 with 0.1mol/L NaOH solution, adding alkaline protease Alcalase 2.4L FG (2.4AU) at a volume ratio of 1.0-2.0mL/L when the temperature reaches 50-60 deg.C, and performing enzymolysis for 120 min; inactivating enzyme at 100 deg.C for 15 min; cooling, centrifuging at 10000r/min for 10min, and collecting supernatant.
(3) Method for obtaining oat antihypertensive polypeptide
Separating the supernatant obtained in the step (2) by a membrane ultrafiltration method, and collecting peptide fragments with Molecular Weight (MW) <5 kDa; and (4) carrying out vacuum freeze drying to obtain powdery oat antihypertensive polypeptide.
The collected powdery oat antihypertensive polypeptide is stored at the temperature of-20 ℃, and is dissolved by normal saline to be prepared into the required concentration when in use.
Example 2
A method for preparing oat antihypertensive polypeptide, comprising the following steps:
(1) extraction of oat protein
Crushing oat grains, sieving the crushed oat grains with a 60-mesh sieve, and mixing the crushed oat grains with a feed-liquid ratio of 1: 9, adding water for dissolving; adjusting pH to 10.0 with 1mol/L NaOH, leaching at constant temperature in 50 deg.C water bath for 120min, centrifuging at 4500r/min for 15min, and separating supernatant (i.e. removing precipitate); adjusting the pH of the supernatant to 4.0 (protein isoelectric point) with 1mol/L HCl, centrifuging at 4500r/min for 15min, and collecting protein precipitate (i.e. removing supernatant); and (3) carrying out vacuum freeze drying on the protein precipitate to obtain oat protein dry powder.
(2) Enzymolysis
Preparing the oat protein dry powder obtained in the step (1) into an aqueous solution with the mass concentration of 8%; placing in a constant temperature water bath kettle, mixing with an electric stirrer, adjusting pH to 9 with 0.1mol/L NaOH solution, adding alkaline protease Alcalase 2.4L FG (2.4AU) at a volume ratio of 1.5-2.0mL/L when the temperature reaches 50-60 deg.C, and performing enzymolysis for 90 min; inactivating enzyme at 100 deg.C for 15 min; cooling, centrifuging at 10000r/min for 10min, and collecting supernatant.
(3) Method for obtaining oat antihypertensive polypeptide
Separating the supernatant obtained in the step (2) by a membrane ultrafiltration method, and collecting peptide fragments with Molecular Weight (MW) <5 kDa; and (4) carrying out vacuum freeze drying to obtain powdery oat antihypertensive polypeptide.
The collected powdery oat antihypertensive polypeptide is stored at the temperature of-20 ℃, and is dissolved by normal saline to be prepared into the required concentration when in use.
Example 3
A method for preparing oat antihypertensive polypeptide, comprising the following steps:
(1) extraction of oat protein
Crushing oat grains, sieving the crushed oat grains with a 60-mesh sieve, and mixing the crushed oat grains with a feed-liquid ratio of 1: 9, adding water for dissolving; adjusting pH to 10.0 with 1mol/L NaOH, leaching at constant temperature in 50 deg.C water bath for 120min, centrifuging at 4500r/min for 15min, and separating supernatant (i.e. removing precipitate); adjusting the pH of the supernatant to 4.0 (protein isoelectric point) with 1mol/LHCl, centrifuging at 4500r/min for 15min, and collecting protein precipitate (i.e. removing supernatant); and (3) carrying out vacuum freeze drying on the protein precipitate to obtain oat protein dry powder.
(2) Enzymolysis
Preparing the oat protein dry powder obtained in the step (1) into an aqueous solution with the mass concentration of 9%; placing in a constant temperature water bath kettle, mixing with an electric stirrer, adjusting pH to 10 with 0.1mol/L NaOH solution, adding alkaline protease Alcalase 2.4L FG (2.4AU) at a volume ratio of 1.5-2.0mL/L when the temperature reaches 50-60 deg.C, and performing enzymolysis for 150 min; inactivating enzyme at 100 deg.C for 15 min; cooling, centrifuging at 10000r/min for 10min, and collecting supernatant.
(3) Method for obtaining oat antihypertensive polypeptide
Separating the supernatant obtained in the step (2) by a membrane ultrafiltration method, and collecting peptide fragments with Molecular Weight (MW) <5 kDa; and (4) carrying out vacuum freeze drying to obtain powdery oat antihypertensive polypeptide.
The collected powdery oat antihypertensive polypeptide is stored at the temperature of-20 ℃, and is dissolved by normal saline to be prepared into the required concentration when in use.
The antihypertensive activity (ACE enzyme inhibitory activity) of the oat polypeptide prepared above was examined by in vitro experiments as follows.
TABLE 1 ACE inhibitory Activity of oat polypeptide samples
| Sample (I) | Inhibition rate of ACE |
| Example 1 | 66.46％ |
| Example 2 | 61.35％ |
| Example 3 | 59.20％ |
In vitro experiment results show that (as shown in table 1), the small molecular peptide obtained by enzymolysis and ultrafiltration of oat protein by alkaline protease can effectively inhibit ACE activity.
The polypeptide prepared by the optimal process is selected to carry out animal experiments so as to concretely show the blood pressure reducing effect of the invention.
Animal grouping and administration mode
The test was carried out using 14-17 week old male SHR rats, weighing 280 + -20 g, supplied by the laboratory animal technology Co., Ltd, of Wei Tong Li Hua, Beijing. Producing license numbers: SCXK (Jing) 2016-: 11400700244657, 11400700244660. Raising in the barrier environment of experimental animal center of Jiangsu university, using license number: SYXK (su) 2013-: 22 ± 2 ℃, humidity: 40-70%, 12h light and shade alternate illumination, and free intake and drinking. All experiments were approved and approved by the ethics committee on laboratory animals of the university of Jiangsu and were performed according to the instructions.
Single test of hypotensive effect
SHR is subjected to intragastric lavage (DIastoll Blood Pressure, DBP) 0h to 10h after performing adaptive training for 10 days, and determinations are made at intervals of 2h by using ALC-NIBP noninvasive Blood Pressure measurement and analysis system, and the results are shown in FIGS. 1 and 2.
TABLE 2 grouping of single hypotensive experiments and dosing
42-day blood pressure lowering effect test
SHR with SBP of more than 170mmHg is used for 42-day blood pressure reduction effect test, after 10-day adaptive training, the SHR is used for performing gavage test, animal groups and administration dosage are shown in table 3, the gavage time lasts for 6 weeks, times of SBP and DBP are measured every week, and the influence of each test object on the blood pressure of SHR rats is observed, and the results are shown in fig. 3 and 4.
TABLE 342 days antihypertensive experimental grouping and dosing
(1) In times of tests of blood pressure lowering effect, the oat polypeptide can remarkably lower SHR blood pressure, SBP reaches the maximum lowering amplitude of 15.34mmHg (P <0.05) when the stomach is perfused for 4 hours, DBP reaches the maximum lowering amplitude of 13.33mmHg (P <0.05) when the stomach is perfused for 6 hours, and the blood pressure lowering effect is continued until the stomach is perfused for 6 hours.
(2) The oat polypeptide has the effect of reducing SHR blood pressure in a 42-day blood pressure reduction effect test, the SBP of a rat begins to decrease at 2 weeks of gastric lavage of the oat polypeptide, and then continuously decreases, the maximum decrease amplitude is 18.84mmHg, and the decrease is 10.55% (P < 0.05). The rat's DBP continued to decrease from week 3 with a maximum decrease of 17.74mmHg, a decrease of 13.43% (P < 0.05). The continuous gavage low-dose oat polypeptide can obviously reduce the blood pressure of rats, has better blood pressure reduction effect than the gavage high-dose oat polypeptide, and takes effect early.
(3) Oat polypeptides exert hypotensive effects primarily by lowering ET-1(P <0.01), TNF- α (P <0.05), Renin (P <0.05), ANGII (P <0.05) levels and increasing BK (P <0.01) and NO levels.
(4) The long-term oat peptide does not affect the organ indexes (P >0.05) of heart, liver, spleen, lung and kidney of SHR, and has no obvious effect on the health condition of each organ of a rat.
Although the invention has been described in detail with respect to and its specific embodiment, it will be apparent to those skilled in the art that variations or modifications may be made thereto without departing from the spirit of the invention.
Claims (10)
1, A method for preparing oat antihypertensive polypeptide, which is characterized by comprising the following steps:
(1) extraction of oat protein
Pulverizing oat grains, sieving, adding water to dissolve, adjusting pH to 10.0, leaching in constant temperature water bath, centrifuging, separating to obtain supernatant, adjusting pH of the supernatant to isoelectric point of protein, centrifuging to obtain protein precipitate, and drying to obtain oat protein dry powder;
(2) enzymolysis
Preparing the oat protein dry powder prepared in the step (1) into aqueous solution, placing the aqueous solution in a constant-temperature water bath kettle, uniformly mixing the aqueous solution with an electric stirrer, adjusting the pH value to 9-10, adding enzyme for enzymolysis, inactivating the enzyme, cooling, centrifuging, and taking supernatant;
(3) method for obtaining oat antihypertensive polypeptide
And (3) separating the supernatant obtained in the step (2) by a membrane ultrafiltration method, collecting peptide fragments, and drying to obtain the powdery oat antihypertensive polypeptide.
2. The preparation method of the oat antihypertensive polypeptide of claim 1, wherein in step (1), the oat grains are crushed and sieved by a 60-mesh sieve, and are dissolved by adding water according to the feed-liquid ratio of 1: 9; the pH was adjusted to 10.0 using 1mol/L NaOH.
3. The method for preparing the oat antihypertensive polypeptide of claim 1, wherein in the step (1), the leaching in the thermostatic waterbath is performed under the condition of being placed in a 50 ℃ water bath for 120 min; after being leached by constant temperature water bath, the mixture is centrifuged for 15min at 4500 r/min.
4. The method for preparing the oat antihypertensive polypeptide of claim 1, wherein in step (1), the pH of the supernatant is adjusted to 4.0 by 1mol/LHCl so as to reach the isoelectric point of protein; centrifuging at 4500r/min for 15min, and collecting protein precipitate; the drying method is vacuum freeze drying.
5. The method for preparing the oat antihypertensive polypeptide according to claim 1, wherein in the step (2), the oat protein dry powder is prepared into an aqueous solution with a mass concentration of 8% -10%; the pH is adjusted to 9-10 with 0.1mol/L NaOH solution.
6. The method for preparing the oat blood pressure-reducing polypeptide of claim 1, wherein in the step (2), Alcalase 2.4L FG is selected for enzymolysis.
7. The method for preparing the oat antihypertensive polypeptide according to claim 1, wherein in the step (2), the enzymolysis conditions are as follows: when the temperature reaches 50-60 ℃, adding 2.4L FG alkaline protease Alcalase according to the volume ratio of 1.5-2.0mL/L, and performing enzymolysis for 90-150 min; the alkaline protease Alcalase 2.4L FG has an enzyme unit of 2.4 AU.
8. The method for preparing the oat antihypertensive polypeptide of claim 1, wherein in step (2), the enzyme deactivation conditions are as follows: inactivating enzyme at 100 deg.C for 15 min; after cooling, centrifuging for 10min at 10000 r/min.
9. The method for preparing the oat antihypertensive polypeptide of claim 1, wherein in step (3), peptide fragments with molecular weight <5kDa are collected.
10. The method for preparing the oat antihypertensive polypeptide of claim 1, wherein in the step (3), the drying method is vacuum freeze drying; the collected powdery oat antihypertensive polypeptide is stored at the temperature of-20 ℃, and is dissolved by normal saline to be prepared into the required concentration when in use.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111513340A (en) * | 2020-06-12 | 2020-08-11 | 通化天肽生物科技有限公司 | Oat polypeptide extraction method and nutritional product |
| CN112655970A (en) * | 2020-12-16 | 2021-04-16 | 江苏大学 | Preparation method of oat polypeptide ferrous chelate |
| CN115353553A (en) * | 2022-06-27 | 2022-11-18 | 上海理工大学 | CCK secretion promoting peptide targeting calcium sensitive receptor and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101117642A (en) * | 2007-07-31 | 2008-02-06 | 北京工商大学 | Polypeptide capable of lowering blood pressure and method for preparing same |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101117642A (en) * | 2007-07-31 | 2008-02-06 | 北京工商大学 | Polypeptide capable of lowering blood pressure and method for preparing same |
Non-Patent Citations (2)
| Title |
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| 耿静静: "超声辅助酶法制备燕麦蛋白ACE抑制肽的研究", 《中国优秀博硕士学位论文全文数据库（硕士）工程科技I辑》 * |
| 马超月: "燕麦多肽的制备及其降血脂和降血压效果研究", 《中国优秀博硕士学位论文全文数据库（硕士） 工程科技I辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111513340A (en) * | 2020-06-12 | 2020-08-11 | 通化天肽生物科技有限公司 | Oat polypeptide extraction method and nutritional product |
| CN112655970A (en) * | 2020-12-16 | 2021-04-16 | 江苏大学 | Preparation method of oat polypeptide ferrous chelate |
| CN115353553A (en) * | 2022-06-27 | 2022-11-18 | 上海理工大学 | CCK secretion promoting peptide targeting calcium sensitive receptor and preparation method and application thereof |
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