CN108003231A - A kind of method of free aminoacid content in reduction small-molecular peptides - Google Patents
A kind of method of free aminoacid content in reduction small-molecular peptides Download PDFInfo
- Publication number
- CN108003231A CN108003231A CN201711111558.7A CN201711111558A CN108003231A CN 108003231 A CN108003231 A CN 108003231A CN 201711111558 A CN201711111558 A CN 201711111558A CN 108003231 A CN108003231 A CN 108003231A
- Authority
- CN
- China
- Prior art keywords
- small
- molecular peptides
- aminoacid content
- free aminoacid
- enzymolysis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
-
- 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
A kind of method for reducing free aminoacid content in small-molecular peptides of the present invention, is related to enzyme process and prepares peptide art, the method for particularly relating to free aminoacid content in a kind of small-molecular peptides for reducing enzymatic isolation method and preparing.The raw material of proteolysis is mostly water-soluble poor albumen, when protease digests insoluble protein body from outside to inside, differ to the Degree of Enzymatic Hydrolysis of the inside and outside layer albumen of protein body, wherein being the major reason of excessive free amino acid to the excessive enzymolysis of particle outer layer albumen.The present invention carries out solubilized pretreatment to protein raw materials, reduces the granular size of albumen, and then plays the role of reducing free amino acid in small-molecular peptides product and generate.
Description
Technical field
The present invention relates to enzyme process to prepare peptide art, particularly relates to dissociate in a kind of small-molecular peptides for reducing enzymatic isolation method and preparing
The method of amino acid content.
Background technology
Small-molecular peptides be a-amino acid with the compound that peptide bond links together and is formed, molecular weight is between protein and ammonia
Between base acid, generally it is formed by connecting by 2~10 amino acid by peptide bond dehydrating condensation.Preparation of industrialization small-molecular peptides master at present
If by protease hydrolyzed approach, the processes such as proteolysis, enzyme deactivation, UF membrane, concentration and spray drying are contained.To improve
Protein transform rate and small-molecular peptides yield, often by the way of excessively digesting, though the method can effectively improve polypeptide yield,
But a large amount of free amino acids can be also generated in enzymolysis product, it might even be possible to more than the 50% of total content.
Amino acid is the base unit for forming small-molecular peptides, but still presence is very big between free amino acid and small-molecular peptides
Difference, including biological activity, infiltration rate and mouthfeel etc., in terms of biological activity, free amino acid can not show a candle to small molecule
Peptide, small-molecular peptides are in addition to the trophism with amino acid, also with blood pressure lowering, antifatigue, raising immunity and other effects；
In terms of absorption rate, the absorptivity of small-molecular peptides is tens times of amino acid.Therefore the free ammonia in separation small-molecular peptides product
Base acid, it is significant to the biological activity of raising small-molecular peptides product.
Existing frequently-used separation method is gel permeation chromatography, this method be mainly using molecular size difference to sample into
Row separation, and free amino acid and small-molecular peptides particularly dipeptides, the molecular size range of tripeptides are close, therefore gel permeation chromatography
It is not ideal enough to the separating effect of free amino acid and small-molecular peptides, while cost is higher, is unfavorable for industrialization amplification.
Free amino acid Producing reason, one of importance are：The raw material of proteolysis is mostly water-soluble poor
Albumen, when protease digests insoluble protein body from outside to inside, to the Degree of Enzymatic Hydrolysis of the inside and outside layer albumen of protein body not
One, wherein the main reason for excessive enzymolysis to particle outer layer albumen is excessive free amino acid.For this reason, from optimization proteolysis
From the point of view of reaction system, protein raw materials are carried out solubilized pre- place by the angle research particularly from protein raw materials pretreatment
Reason, reduces the granular size of albumen, is generated with having the function that to reduce free amino acid in small-molecular peptides product, grinding in this respect
Study carefully there is not yet reporting.
The content of the invention
The purpose of the invention is to overcome the shortcomings of conventional method, there is provided one kind reduces free amino acid in small-molecular peptides
The method of content.
In order to realize foregoing invention purpose, technical solution is as follows：Free aminoacid content in a kind of reduction small-molecular peptides
Method, carries out as steps described below：
A certain amount of protein raw materials are taken, solubilized pretreatment is carried out, is completely dissolved it, at peak enzymolysis-ability temperature and pH,
Add protease to be digested, enzymolysis process maintains optimum temperature and pH, waits after digesting, enzyme deactivation, centrifuging and taking supernatant, ultrafiltration
UF membrane takes filtered solution, dry.
Wherein described albumen includes and is not limited to casein and zeins.
It is wherein described to be included with solubilized preprocessing means and be not limited to adjust solvent polarity, temperature or pH.
Wherein described protease includes and is not limited to alkali protease, neutral proteinase, pancreatin and flavor protease.
Wherein described casein small-molecular peptides preparation method is：
Casein is taken, pH to 8.0 is adjusted with NaOH, after being completely dissolved it, under the conditions of 50 DEG C and pH 7.0, in addition
Property protease digested, enzymolysis process maintains temperature and pH constant, digests enzyme deactivation after 8-12h, centrifuging and taking supernatant, ultrafiltration membrane point
From filtered solution is taken, dry.
Wherein described zeins small-molecular peptides preparation method, carries out as steps described below：
Zeins is taken, is dissolved with 60% ethanol solution of certain volume, at the uniform velocity agitating solution is until it is complete molten, slowly
Add a certain amount of water make concentration of alcohol be diluted to 20% after (final concentration of protein 1.5% (m/v)), in 50 DEG C and 8.0 conditions of pH
Under, add pancreatin and digested (by enzyme bottom than 5% (m/m)), enzymolysis process maintains temperature and pH constant, digests 9-16h, adjusts
PH to 4-5, after revolving removes residual ethanol, moisturizing enzyme deactivation, centrifuging and taking supernatant, Ultra filtration membrane takes filtered solution, dry.
Advantages of the present invention：
Using solubilized pretreatment, free aminoacid content in small-molecular peptides is effectively reduced, reduced rate reaches 21.3%~
72.9%.
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 simply further described the present invention, 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.
Neutral proteinase used in the present embodiment and reference examples and pancreatin purchase believe the limited public affairs of (China) biotechnology from Novi
Department.Neutral proteinase enzyme activity 2.01 × 105U/mL, pancreatin enzyme activity 1.09 × 105U/g。
Casein used in the present embodiment and reference examples is purchased from mountain purchased from Shanghai is more than Industrial Co., Ltd., zeins
Eastern day is into bio tech ltd.
The present embodiment and reference examples free amino acid detection method are as follows：
Take the sample 5mL after enzyme deactivation to add 7% sulfosalicylic acid 5mL to be uniformly mixed, (4000r/ is centrifuged after standing 30min
Min, 5min) take 4mL supernatants to cross 3kD ultra-filtration centrifuge tubes, filtered solution is transferred to 50mL after centrifugation (4000r/min, 30min)
Volumetric flask constant volume, sample solution retain the standby survey of filtrate after crossing 0.22 μm of miillpore filter.According to method as defined in GB/T5009.124,
Its free aminoacid content is measured with automatic amino acid analyzer.
Reference examples 1
500mL beakers are taken, add 12g caseins and 400mL water, are added under conditions of hydrolysis temperature is 50 DEG C, pH is 7.0
Enter 600 μ L (enzyme activity 2.01 × 10 of neutral proteinase5U/mL), maintain temperature and pH constant in enzymolysis process, digest beaker after 8h
It is placed in 10min enzyme deactivations in boiling water bath.The free aminoacid content of enzymolysis product is 7.71 ± 0.58mg/mL.
Reference examples 2
500mL beakers are taken, add 6.75g zeins and 450mL water.In the condition that hydrolysis temperature is 50 DEG C, pH is 8.0
Lower addition pancreatin 337.5mg (enzyme activity 1.09 × 105U/g), maintain temperature and pH constant in enzymolysis process, adjusted after digesting 9h
pH.The free aminoacid content of enzymolysis product is 4.36 ± 0.53mg/mL.
Embodiment 1
500mL beakers are taken, add 12g caseins and 400mL water, solubilized pretreatment is carried out after being stirred with glass bar and (is adjusted
Section pH to 8.0 is simultaneously kept, until casein is entirely molten).Neutral protein is added under conditions of hydrolysis temperature is 50 DEG C, pH is 7.0
600 μ L (enzyme activity 2.01 × 10 of enzyme5U/mL), maintain temperature and pH constant in enzymolysis process, beaker is placed in boiling water bath after digesting 8h
Middle 10min enzyme deactivations.The free aminoacid content of enzymolysis product is 5.21 ± 0.49mg/mL.
Embodiment 2
500mL beakers are taken, add 12g caseins and 400mL water, solubilized pretreatment is carried out after being stirred with glass bar and (is adjusted
Section pH to 8.0 is simultaneously kept, until casein is entirely molten).Neutral protein is added under conditions of hydrolysis temperature is 50 DEG C, pH is 7.0
600 μ L (enzyme activity 2.01 × 10 of enzyme5U/mL), maintain temperature and pH constant in enzymolysis process, beaker is placed in boiling water bath after digesting 12h
Middle 10min enzyme deactivations.The free aminoacid content of enzymolysis product is 6.07 ± 0.42mg/mL.
Embodiment 3
500mL beakers are taken, it is (molten with 60% ethanol solution 150mL that solubilized pretreatment is carried out after addition 6.75g zeins
Zein, at the uniform velocity agitating solution are solved until it is complete molten, being slowly added to water 300mL makes concentration of alcohol be diluted to 20%).Digesting
Pancreatin 337.5mg (enzyme activity 1.09 × 10 is added under conditions of temperature is 50 DEG C, pH is 8.05U/g), temperature is maintained in enzymolysis process
Degree and pH are constant, and beaker is placed in 10min enzyme deactivations in boiling water bath after digesting 9h.The free aminoacid content of enzymolysis product for 1.18 ±
0.45mg/mL。
Embodiment 4
500mL beakers are taken, it is (molten with 60% ethanol solution 150mL that solubilized pretreatment is carried out after addition 6.75g zeins
Zein, at the uniform velocity agitating solution are solved until it is complete molten, being slowly added to water 300mL makes concentration of alcohol be diluted to 20%).Digesting
Pancreatin 337.5mg (enzyme activity 1.09 × 10 is added under conditions of temperature is 50 DEG C, pH is 8.05U/g), temperature is maintained in enzymolysis process
Degree and pH are constant, and beaker is placed in 10min enzyme deactivations in boiling water bath after digesting 16h.The free aminoacid content of enzymolysis product is 2.33
±0.56mg/mL。
Claims (6)
- A kind of 1. method for reducing free aminoacid content in small-molecular peptides, it is characterised in that carry out as steps described below：Take one Quantitative Western raw material, carries out solubilized pretreatment, is completely dissolved it, at peak enzymolysis-ability temperature and pH, add protease into Row enzymolysis, enzymolysis process maintain optimum temperature and pH, wait after digesting, enzyme deactivation, centrifuging and taking supernatant, Ultra filtration membrane takes filtration Liquid, it is dry.
- A kind of 2. method for reducing free aminoacid content in small-molecular peptides according to claim 1, it is characterised in that bag Include and be not limited to casein and zeins.
- A kind of 3. method for reducing free aminoacid content in small-molecular peptides according to claim 1, it is characterised in that bag Include and be not limited to adjust solvent polarity, temperature and pH.
- A kind of 4. method for reducing free aminoacid content in small-molecular peptides according to claim 1, it is characterised in that bag Include and be not limited to alkali protease, neutral proteinase, pancreatin, flavor protease.
- 5. a kind of method for reducing free aminoacid content in small-molecular peptides according to claim 1, it is characterised in that take Casein, pH to 8.0 is adjusted with NaOH, after being completely dissolved it, under the conditions of 50 DEG C and pH 7.0, add neutral proteinase into Row enzymolysis, enzymolysis process maintain temperature and pH constant, digest enzyme deactivation after 8-12h, centrifuging and taking supernatant, and Ultra filtration membrane takes filtration Liquid, it is dry.
- 6. a kind of method for reducing free aminoacid content in small-molecular peptides according to claim 1, it is characterised in that take Zeins, is dissolved with 60% ethanol solution, and at the uniform velocity for agitating solution until it is complete molten, being slowly added to a certain amount of water makes ethanol Concentration dilution under the conditions of 50 DEG C and pH 8.0, adds pancreatin and is digested to after 20%, and enzymolysis process maintains temperature and pH permanent It is fixed, 9-16h is digested, pH is adjusted to acidity, rotates after removing residual ethanol, moisturizing enzyme deactivation, centrifuging and taking supernatant, Ultra filtration membrane takes Filtered solution, it is dry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711111558.7A CN108003231A (en) | 2017-11-13 | 2017-11-13 | A kind of method of free aminoacid content in reduction small-molecular peptides |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711111558.7A CN108003231A (en) | 2017-11-13 | 2017-11-13 | A kind of method of free aminoacid content in reduction small-molecular peptides |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108003231A true CN108003231A (en) | 2018-05-08 |
Family
ID=62051432
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711111558.7A Pending CN108003231A (en) | 2017-11-13 | 2017-11-13 | A kind of method of free aminoacid content in reduction small-molecular peptides |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108003231A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1833031A (en) * | 2003-08-01 | 2006-09-13 | 卡尔皮斯株式会社 | Casein hydrolyzate, process for producing the same and use thereof |
| CN102787155A (en) * | 2012-08-30 | 2012-11-21 | 甘肃华羚生物技术研究中心 | Preparation method of yak milk casein antihypertensive peptide |
| CN103352064A (en) * | 2013-06-25 | 2013-10-16 | 天津大学 | Method for preparing corn protein active peptide by using composite carrier immobilized double enzymes |
| CN103923964A (en) * | 2014-04-17 | 2014-07-16 | 江南大学 | Method for preparing casein hydrolysate with stable functionality by using different hydrolysis conditions |
| CN107163133A (en) * | 2017-06-26 | 2017-09-15 | 大连工业大学 | A kind of biologically active peptide and preparation method thereof |
-
2017
- 2017-11-13 CN CN201711111558.7A patent/CN108003231A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1833031A (en) * | 2003-08-01 | 2006-09-13 | 卡尔皮斯株式会社 | Casein hydrolyzate, process for producing the same and use thereof |
| CN102787155A (en) * | 2012-08-30 | 2012-11-21 | 甘肃华羚生物技术研究中心 | Preparation method of yak milk casein antihypertensive peptide |
| CN103352064A (en) * | 2013-06-25 | 2013-10-16 | 天津大学 | Method for preparing corn protein active peptide by using composite carrier immobilized double enzymes |
| CN103923964A (en) * | 2014-04-17 | 2014-07-16 | 江南大学 | Method for preparing casein hydrolysate with stable functionality by using different hydrolysis conditions |
| CN107163133A (en) * | 2017-06-26 | 2017-09-15 | 大连工业大学 | A kind of biologically active peptide and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TWI597017B (en) | Method for preparing pearl protein and water-soluble pearl protein and acid-soluble pearl protein prepared by the method | |
| CN105017412B (en) | A method of the separating high-purity bovine serum albumin(BSA) from cow's serum | |
| CN102702274B (en) | Method for preparing high-purity soybean oligosaccharide from soybean whey wastewater | |
| CN105331665B (en) | A kind of method that pig brain proteolysis prepares brain polypeptide and brain small-molecular peptides | |
| CN106947798A (en) | A kind of preparation method of chickpea peptides | |
| CN105524966A (en) | Method for preparing ACE inhibitory peptides through bean pulp enzymolysis | |
| CN110272935A (en) | A kind of peanut antioxidant peptide and preparation method thereof of high pressure assistance enzymolysis preparation | |
| CN109320588A (en) | A kind of ACE inhibitory activity peptide in stichopus japonicus source | |
| CN108378364A (en) | It is a kind of water solubility maize oligopeptide/curcumin composite nanometer particle and its solution preparation method | |
| CN110283869B (en) | Preparation method of 7-aminocephalosporanic acid | |
| US9534026B2 (en) | Corn active peptide additive for cell culture medium | |
| CN103740797B (en) | Method for preparing high-hydrolysis degree functional oligopeptide by use of high-temperature peanut meal | |
| CN106701877A (en) | Method for preparing ACE inhibitory peptide derived from oysters | |
| CN112111546B (en) | Industrial preparation method of phosvitin phosphopeptide and vitellin polypeptide | |
| CN104877007B (en) | Yak milk lactalbumin source ACE inhibitor peptides and preparation method thereof | |
| CN108003231A (en) | A kind of method of free aminoacid content in reduction small-molecular peptides | |
| CN110144376A (en) | Nanoscale collagen peptide and preparation method thereof | |
| CN105085651B (en) | A kind of casein phosphopeptide monomer and preparation method thereof | |
| CN102108348A (en) | Method for extracting papain | |
| CN108191991A (en) | A kind of method of purification of Polysaccharide in Pleurotus eryngii | |
| CN109956950A (en) | A kind of joint production process extracting Hemoglobin Peptide and ferroheme from pig blood | |
| CN107082796A (en) | A kind of method of purifying protein zymolyte small molecular polypeptide | |
| CN101560508A (en) | High-purity papain extraction method | |
| CN1166688C (en) | Method of separating lactotransferrin from whey liquid | |
| CN110105444A (en) | A kind of preparation method of controllable molecular weight Isin glue collagen |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180508 |