CN108003230A - A kind of method for preparing zeins blood pressure lowering peptide - Google Patents
A kind of method for preparing zeins blood pressure lowering peptide Download PDFInfo
- Publication number
- CN108003230A CN108003230A CN201711111333.1A CN201711111333A CN108003230A CN 108003230 A CN108003230 A CN 108003230A CN 201711111333 A CN201711111333 A CN 201711111333A CN 108003230 A CN108003230 A CN 108003230A
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- Prior art keywords
- zeins
- blood pressure
- pressure lowering
- pancreatin
- preparing
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
- C07K14/425—Zeins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Abstract
The invention discloses a kind of method for preparing zeins blood pressure lowering peptide, it is related to processing of agriculture product field.More particularly to a kind of under the alcoholic environment of low concentration, successively using the method for pancreatin and pepsin enzymolysis zeins preparation high activity blood pressure lowering peptide.The problem of dissolubility is poor under water-soluble environment, and enzymolysis efficiency is low the invention avoids zeins.Under low-concentration ethanol environment, pancreatin and pepsin have greater activity, while zeins dissolubility is fine, so as to significantly improve enzymolysis efficiency, greatly improve enzymolysis product hypotensive activity, reduce manufacturing cost.
Description
Technical field
The present invention relates to processing of agriculture product field, and in particular under the molten environment of alcohol, due to pancreatin and pepsin
There is preferable tolerance to ethanol, digest the method that zeins prepares blood pressure lowering peptide successively.
Background technology
Zeins aboundresources.China is maize production big country, 2.15 hundred million tons of corn annual output in 2014.Corn
Main By product in starch production process is corn protein powder, and wherein alcohol soluble protein accounts for 50%~60%.Zeins
Rich in nutrition content, and poisonous and harmful substance is free of, it is extensive currently with the film forming of zeins, inoxidizability
Applied to the field such as packaging, fresh-keeping, but its higher value application degree is low.Recent study shows that zeins is to prepare drop
The ideal protein resource of blood pressure peptide, and zeins blood pressure lowering peptide is prepared using enzymatic isolation method mostly.But corn alcohol
Molten albumen is soluble in ethanol solution, is insoluble in rich in hydrophobic amino acids such as glutamic acid, leucine, proline and phenylalanines
Water, even the extremely strong alkali protease of hydrolysis ability also is difficult to " attack " its restriction enzyme site, not only enzymolysis efficiency is low, but also alkali
Property protease price it is expensive, improve manufacturing cost.
Pancreatin (containing trypsase and chymotrypsin etc.) and pepsin are digestible protein enzymes important in animal body, its
With stronger specificity, the peptide bond that wherein trypsase forms basic amino acid has high specificity, chymotrypsin energy
Single-minded hydrolysis Tyr, Trp and Phe carboxy-terminal peptide bond.The blood pressure lowering peptide structure-activity relationship that Jia Jun is studied by force shows that enzymolysis prepares blood pressure lowering
During peptide, in the protease of Tyr, Pro, Trp, Phe and Leu c-terminus, (the strong ultrasounds of Jia Jun prepare enzyme process small preferred restriction enzyme site
The influence of wheat germ blood pressure lowering peptide and its mechanism of action [D] Jiangsu Universitys, 2009.).Wherein pepsin can be preferentially broken
The peptide bond formed by hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine or leucines.The extraction of pancreatin and pepsin
Technique is simple, is not related to purifying process, and cheap.Under water-soluble environment, zeins dissolubility is poor, stomach cardia
Enzyme, pancreatin are difficult to touch its restriction enzyme site, and its hydrolysis ability is not so good as alkali protease, still, pancreatin and pepsin pair
Ethanol has very strong tolerance, studies it zeins is digested in alcoholic solution and prepare biologically active peptide, yet there are no report
Road.
The content of the invention
The purpose of the invention is to overcome prior art there are the problem of, there is provided one kind under the molten environment of alcohol, utilize stomach
The method that intestinal digestion enzyme (pepsin and pancreatin) enzymolysis zeins prepares blood pressure lowering peptide.Specially in the second of low concentration
In alcohol environment, zeins is digested using pancreatin, pepsin successively, prepares the blood pressure lowering peptide of high activity.
In order to realize foregoing invention purpose, its specific technical solution is as follows：
A kind of method for preparing zeins blood pressure lowering peptide, carries out as steps described below：
By zeins with after the ethanol dissolving of 60%-70%, 35~50 DEG C are adjusted the temperature to, addition contains pancreatin
Aqueous solution digested so that ethanol final concentration is reduced to 15~20%, (m/v) pancreatin of final concentration of protein 1.5% additive amount 3%
~5% (E/S), use NaOH solution maintain pH for 7.5~8.5, digest 3~6h after terminate, add HCl adjust pH be 1.5~
After 2.0, pH stablize, add pepsin and digested, pepsin additive amount 3%~5% (E/S), maintain pH for 1.5~
2.0, after digesting 2~4h, adjust pH to be terminated for alkalescence and react, rotary evaporation removes ethanol, and enzyme deactivation after moisturizing, centrifugation, collects supernatant
Liquid, it is dry.
Advantages of the present invention：Under the molten environment of alcohol, blood pressure lowering is prepared with pancreatin and pepsin enzymolysis zeins
The advantage of peptide, on the one hand, zeins is soluble in 60% ethanol, which is slowly diluted with water to 15~20% second
Alcoholic solution, zeins in a short time will not segregation, be effectively increased the contact area of enzyme and albumen, be more advantageous to
Enzyme " attack " its restriction enzyme site；On the other hand, pancreatin and pepsin have good tolerance, pancreatin and stomach cardia to ethanol
Enzyme is the common protease for preparing blood pressure lowering peptide, and cheap.The present invention is under conditions of proteinase activity is higher, not only
The dissolubility of zeins is effectively improved, and effectively improves the activity of enzymolysis product blood pressure lowering peptide, so as to effectively reduce
Production cost.
Below by embodiment, technical scheme is described in further detail.
Embodiment
Used term in the present invention, it is unless otherwise specified, generally usual with those of ordinary skill in the art
The implication of understanding.The present invention is described in further detail with reference to specific embodiment, and with reference to data.It is to be understood that these
Embodiment is that the present invention is further described, it is impossible to is interpreted as limiting the scope of the present invention, the skill in the field
Art engineer can make the present invention some nonessential modifications and adaptations according to the content of foregoing invention.
Below in an example, the various processes and method not being described in detail are conventional methods as known in the art.
The source of agents useful for same, trade name and it is necessary to list its constituent person, indicates on the first appearance, thereafter phase used
With reagent unless otherwise specified, it is identical with the content indicated first.
The assay method of hypotensive activity is as follows in reference examples and embodiment：
External inhibitory activity quick determination method (the Dingqing sesame of angiotensin transferase (ACE) established according to Dingqing sesame
Impulse ultrasound assistance enzymolysis method prepares research [D] the Jiangsu Universitys of maize yellow-powder ACEI active peptides, and 2008.).
Reference examples 1
Zeins 6.75g is weighed, adds 150mL distilled water, then temperature adjustment weighs 337.5mg to 40 DEG C
Pancreatin suspension is made in pancreatin in 300mL distilled water, is slowly added to pancreatin suspension while stirring, stablizes pH with 1M NaOH
To 8.0, constantly dropwise addition NaOH maintenances pH is constant in enzymolysis process, and bath temperature is constant.Terminate after enzymolysis 4h.Add 1M HCl
PH to 2.0 is adjusted, constantly dropwise addition NaOH maintenances pH is constant in enzymolysis process, and bath temperature is constant.Add the stomach cardia of 270mg
Enzyme digests, and terminates after digesting 4h.Then sample, rapid boiling water bath boils 10min, and after cooling, 10000rpm centrifugation 10min, are received
Collect supernatant, -20 DEG C freeze, ACE inhibitory activity to be measured.The determination experiment of hypotensive activity the result shows that：Enzymolysis product ACE presses down
System activity is 31.2%.
Embodiment 1
Weigh zeins 6.75g, add 70% ethanol solutions of 150mL and be completely dissolved to it, temperature adjustment to 40
DEG C, weigh 337.5mg pancreatin and pancreatin suspension is made in 375mL distilled water, be slowly added to pancreatin suspension while stirring, use
1M NaOH stablize pH to 8.5, and constantly dropwise addition NaOH maintenances pH is constant in enzymolysis process, and bath temperature is constant.Tied after enzymolysis 6h
Beam.Add 1M HCl and adjust pH to 1.5, constantly dropwise addition NaOH maintenances pH is constant in enzymolysis process, and bath temperature is constant.Add
270mg pepsins digest, and terminate after digesting 4h.Adjust pH to terminate for 8 to react, rotary evaporation removes ethanol, boiling water bath after moisturizing
10min is boiled, is cooled down, 10000rpm centrifugation 10min, collect supernatant, -20 DEG C freeze, ACE inhibitory activity to be measured.Blood pressure lowering
Activity determination experiment the result shows that：Enzymolysis product ACE inhibitory activity is 82.7%.
Embodiment 2
Weigh zeins 6.75g, add 60% ethanol solutions of 150mL and be completely dissolved to it, temperature adjustment to 40
DEG C, weigh 337.5mg pancreatin and pancreatin suspension is made in 300mL distilled water, be slowly added to pancreatin suspension while stirring, use
1M NaOH stablize pH to 7.5, and constantly dropwise addition NaOH maintenances pH is constant in enzymolysis process, and bath temperature is constant.Tied after enzymolysis 3h
Beam.Add 1M HCl and adjust pH to 2.0, constantly dropwise addition NaOH maintenances pH is constant in enzymolysis process, and bath temperature is constant.Add
270mg pepsins digest, and terminate after digesting 2h.Adjust pH to terminate for 8 to react, rotary evaporation removes ethanol, boiling water bath after moisturizing
10min is boiled, is cooled down, 10000rpm centrifugation 10min, collect supernatant, -20 DEG C freeze, ACE inhibitory activity to be measured.Blood pressure lowering
Activity determination experiment the result shows that：Enzymolysis product ACE inhibitory activity is 75.4%.
Embodiment 3
Weigh zeins 6.75g, add 60% ethanol solutions of 150mL and be completely dissolved to it, temperature adjustment to 35
DEG C, weigh 202.5mg pancreatin and pancreatin suspension is made in 300mL distilled water, be slowly added to pancreatin suspension while stirring, use
1M NaOH stablize pH to 8.0, and constantly dropwise addition NaOH maintenances pH is constant in enzymolysis process, and bath temperature is constant.Tied after enzymolysis 4h
Beam.Add 1M HCl and adjust pH to 2.0, constantly dropwise addition NaOH maintenances pH is constant in enzymolysis process, and bath temperature is constant.Add
202.5mg pepsins digest, and terminate after digesting 2h.Adjust pH to terminate for 8 to react, rotary evaporation removes ethanol, boiling water after moisturizing
10min is boiled in bath, cools down, and 10000rpm centrifugation 10min, collect supernatant, -20 DEG C freeze, ACE inhibitory activity to be measured.Blood drops
Press activity determination experiment the result shows that：Enzymolysis product ACE inhibitory activity is 63.8%.
Embodiment 4
Weigh zeins 6.75g, add 60% ethanol solutions of 150mL and be completely dissolved to it, temperature adjustment to 50
DEG C, weigh 202.5mg pancreatin and pancreatin suspension is made in 450mL distilled water, be slowly added to pancreatin suspension while stirring, use
1M NaOH stablize pH to 7.5, and constantly dropwise addition NaOH maintenances pH is constant in enzymolysis process, and bath temperature is constant.Tied after enzymolysis 3h
Beam.Add 1M HCl and adjust pH to 2.0, constantly dropwise addition NaOH maintenances pH is constant in enzymolysis process, and bath temperature is constant.Add
The pepsin enzymolysis of 202.5mg, terminates after digesting 2h.Adjust pH to terminate for 8 to react, rotary evaporation removes ethanol, boils after moisturizing
10min is boiled in water-bath, cooling, and 10000rpm centrifugation 10min, collect supernatant, -20 DEG C freeze, ACE inhibitory activity to be measured.Drop
The determination experiment of blood pressure activity the result shows that：Enzymolysis product ACE inhibitory activity is 54.3%.
Claims (5)
- A kind of 1. method for preparing zeins blood pressure lowering peptide, it is characterised in that：Zeins is carried out solubilized Processing, adjusts the temperature to 30~50 DEG C, adds the aqueous solution containing pancreatin, digested, and it is 7.0~8.5 to maintain pH with lye, For enzymolysis to after a certain period of time, adding acid makes its pH to 1.5~2.0, adds pepsin and is digested, maintain pH for 1.5~ 2.0, digest to reaction is terminated after a certain period of time, remove ethanol, moisturizing enzyme deactivation, cools down, dry, is prepared into Semen Maydis polypeptide finished product.
- 2. a kind of method for preparing zeins blood pressure lowering peptide according to claim 1, it is characterised in that use 60%~70% ethanol is completely dissolved zein.
- 3. a kind of method for preparing zeins blood pressure lowering peptide according to claim 1, it is characterised in that add pancreas After the aqueous solution of enzyme so that concentration of alcohol drops to 15~20%.
- 4. a kind of method for preparing zeins blood pressure lowering peptide according to claim 1, it is characterised in that pancreatin adds Dosage 3%~5% (E/S), pepsin additive amount 3%~5% (E/S).
- A kind of 5. method for preparing zeins blood pressure lowering peptide according to claim 1, it is characterised in that pancreatin enzyme Solve 3~6h of time, 2~4h of pepsin enzymolysis time.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711111333.1A CN108003230B (en) | 2017-11-13 | 2017-11-13 | Method for preparing zein antihypertensive peptide |
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| CN201711111333.1A CN108003230B (en) | 2017-11-13 | 2017-11-13 | Method for preparing zein antihypertensive peptide |
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| CN108003230B CN108003230B (en) | 2020-11-03 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114656548A (en) * | 2022-04-13 | 2022-06-24 | 练文飞 | Antihypertensive peptide, and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1661031A (en) * | 2004-12-22 | 2005-08-31 | 华南理工大学 | Method for enzyme hydrolyzing zein through two steps method |
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2017
- 2017-11-13 CN CN201711111333.1A patent/CN108003230B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1661031A (en) * | 2004-12-22 | 2005-08-31 | 华南理工大学 | Method for enzyme hydrolyzing zein through two steps method |
Non-Patent Citations (5)
| Title |
|---|
| Y MURAKAMI: "Novel Process for Enzymatic Hydrolysis of Proteins in an Aqueous Two-Phase System for the Production of Peptide Mixture", 《PREP BIOCHEM BIOTECHNOL》 * |
| 刘志国，等: "玉米醇溶蛋白酶解制备ACE抑制肽的研究", 《食品研究与开发》 * |
| 刘良忠等: "双相溶剂中分步水解玉米醇溶蛋白制备玉米肽的研究", 《第十四届国际谷物科技与面包大会暨国际油料与油脂发展论坛》 * |
| 崔兆惠； 李书国；: "水-乙醇二元体系中酶解法制备玉米活性肽", 《粮食科技与经济 》 * |
| 江莉莉: "玉米肽的制备及其生理活性的初步研究", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114656548A (en) * | 2022-04-13 | 2022-06-24 | 练文飞 | Antihypertensive peptide, and preparation method and application thereof |
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Inventor after: Ma Haile Inventor after: Huang Panfen Inventor after: Li Yunliang Inventor after: Ruan Siyu Inventor after: Wang Yucheng Inventor after: Yang Xue Inventor after: Lu Feng Inventor after: Huang Chang Inventor before: Huang Panfen Inventor before: Ma Haile Inventor before: Li Yunliang Inventor before: Ruan Siyu Inventor before: Wang Yucheng Inventor before: Yang Xue Inventor before: Lu Feng Inventor before: Huang Chang |
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