CN108251482A - The production method of three enzymes, one ferment chicken embryo peptide - Google Patents
The production method of three enzymes, one ferment chicken embryo peptide Download PDFInfo
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- CN108251482A CN108251482A CN201810167938.0A CN201810167938A CN108251482A CN 108251482 A CN108251482 A CN 108251482A CN 201810167938 A CN201810167938 A CN 201810167938A CN 108251482 A CN108251482 A CN 108251482A
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- 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
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- 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
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Abstract
The production method of three enzymes, one ferment chicken embryo peptide provided by the invention first passes around ptyalin enzymolysis, first starch-splitting etc., in order to avoid influencing pepsin and trypsase to the enzymolysis of protein in chicken embryo etc., improves to obtain peptide rate.Then hydrolysis temperature, pH environment and enzymolysis time of pepsin and trypsase etc. is controlled, improve enzymolysis efficiency and ensures that peptide molecular weight is smaller.Streptomycete fermentation is carried out after 3 enzymolysis, small peptide is further broken into, makes the chicken embryo peptide molecular weight smaller of preparation, it is easier to by people to absorb.Then by separation, ultrafiltration is redissolved, column chromatography after concentration, and obtained Product Activity peptide pick-up rate is high, loss amount<1%, relative molecular mass is less than the small-molecular peptides of 2KD>91%.Compared with prior art, the method that the present invention uses bionic enzymatic by digesting three times and one time fermentation, makes that chicken embryo enzymolysis is complete, and obtained chicken embryo peptide molecule smaller, yield is more, is more advantageous to absorption of human body, plays its effect.
Description
Technical field
The present invention relates to the production methods of chicken embryo peptide, and in particular to the production method of three enzymes, one ferment chicken embryo peptide utilizes successively
Ptyalin, pepsin and trypsin digestion, finally using streptomycete fermentation, whole process bionic enzymatic obtains chicken
Embryo peptide.
Background technology
The amino of one amino acid can be condensed into peptide with the carboxyl of another amino acid, and the amide groups of formation is in protein
It is known as peptide bond in chemistry.The molecule of amino acid is minimum, and protein is maximum, and two or more amino acid dehydrating condensations forms several
As soon as a peptide bond, so as to form a peptide, multiple peptides carry out multistage fold and form a protein molecule.
The embryo of chicken embryo, that is, chicken is that fertilized eggs are hatched to certain number of days, the edible egg of also non-broken shell.Study table
It is bright, the nutritional ingredient included in chicken embryo include protein, polypeptide, amino acid, carbohydrate (glucose, fructose, galactolipin,
Arabinose, furanose etc.), lipid, aliphatic acid etc..In addition, anti-infective ingredient and bioactive substance are further included in chicken embryo.Institute
The anti-infective ingredient stated has globulin, interferon, fetoprotein, lysozyme and phosphodiesterase；The bioactive substance
Have sialic acid, youngster's phenol amine, insulin like growth factor-1, insulin, kininase, histaminase, choline, erythropoietin(EPO),
Deoxyribonuclease and ribalgilase etc..Relative to unhatched egg, nutritional ingredient, particularly small molecule egg in chicken embryo
White or polypeptide content is more abundant, and then declines to a great extent to fat, the cholesterol equal size that human body is paid no attention to.
Invention content
The purpose of the present invention is to provide a kind of production method of three enzymes, one ferment chicken embryo peptide, successively using ptyalin,
Pepsin and trypsin digestion finally utilize streptomycete fermentation, whole process bionic enzymatic, obtained chicken embryo peptide activity
Height, total peptide content is high, and degree of safety is high.
The production method of three enzymes, one ferment chicken embryo peptide provided by the invention, includes the following steps：
1) chicken embryo hatched 8-15 days is taken out, is crushed, after water is added to be homogenized, pH to 6.5-7.5 is adjusted, adds in ptyalase
Enzyme carries out enzyme digestion reaction under 37-37.5 DEG C of heat-retaining condition；
2) and then pH to 1.5-2.0 is adjusted, adds in pepsin, enzyme digestion reaction is carried out under 37-42 DEG C of heat-retaining condition；
3) and then pH to 7.5-8.0 is adjusted, adds in trypsase, enzyme digestion reaction is carried out under 37-42 DEG C of heat-retaining condition；
4) streptomycete is added in into system made from step 3), after fermentation, centrifugation, filtrate ultrafiltration after separation, obtained filter
Liquid is dried in vacuo；
5) step 4) desciccate is dissolved in water, and then carries out column chromatography, is then concentrated under reduced pressure, dry to get chicken
Embryo peptide.
Described in step 1) plus water homogenate refers to add in the water of 1-3 times of chicken embryo quality.
The addition of ptyalin is the 0.6-5.5% of chicken embryo quality in step 1).
Enzyme digestion reaction described in step 1) carries out 1-2h；
The addition of pepsin is the 0.3-3.5% of chicken embryo quality in step 2).
Enzyme digestion reaction described in step 2) carries out 2-4h；
The addition of trypsase is the 0.3-3% of chicken embryo quality in step 3).
Enzyme digestion reaction described in step 3) carries out 3-5h；
The addition of step 4) Streptomyces is the 0.05-1% of chicken embryo quality.
Fermentation condition is 30-37 DEG C of fermentation 20-30h in step 4).
Centrifugation described in step 4) refers to centrifuge 15-20min under the conditions of 8000-10000r/min.
Affiliated ultrafiltration refers to carry out ultrafiltration with the hollow-fibre membrane that retention relative molecular mass is 3KD in step 4).
It is dissolved in water described in step 5) and refers to the water dissolution for adding 3-5 times.
Column chromatography described in step 5) refers to, using DA-201C type macroporous resin column chromatographies, after being eluted with water, then use
Ethanol elution collects de- liquid.
The present invention first passes around ptyalin enzymolysis, first starch-splitting etc., in order to avoid influence pepsin and trypsase
To the enzymolysis of protein in chicken embryo etc., peptide rate is improved to obtain.Then hydrolysis temperature, the pH environment of pepsin and trypsase are controlled
And enzymolysis time etc., it improves enzymolysis efficiency and ensures that peptide molecular weight is smaller.Streptomycete fermentation is carried out after 3 enzymolysis, into
One step is decomposed into small peptide, makes the chicken embryo peptide molecular weight smaller of preparation, solubility higher, it is easier to by people to absorb.Then pass through
Separation, ultrafiltration are redissolved, column chromatography after concentration, and obtained Product Activity peptide pick-up rate is high, loss amount<1%, opposite point
Protonatomic mass is less than the small-molecular peptides of 2KD>91%.
Compared with prior art, the present invention use bionic enzymatic method, successively using ptyalin, pepsin and
Trypsin digestion finally utilizes streptomycete fermentation.By digesting three times and one time fermentation, make chicken embryo enzymolysis complete, obtain
Chicken embryo peptide molecule smaller, yield is more, is more advantageous to absorption of human body, plays its effect.
Specific embodiment
Embodiment 1
A kind of production method of three enzymes, one ferment chicken embryo peptide, includes the following steps：
1) chicken embryo hatched 10 days is taken out, is crushed, after the water homogenate for adding 2 times of amounts, pH to 7 is adjusted, adds in chicken embryo quality 1%
Ptyalin, enzyme digestion reaction 2h is carried out under 37.5 DEG C of heat-retaining conditions；
2) and then pH to 1.5 is adjusted, adds in the pepsin of chicken embryo quality 0.5%, enzyme is carried out under 38 DEG C of heat-retaining conditions
Solution reaction 4h；
3) and then pH to 7.5 is adjusted, adds in the trypsase of chicken embryo quality 0.5%, enzyme is carried out under 38 DEG C of heat-retaining conditions
Solution reaction 5h；
4) streptomycete of chicken embryo quality 0.08% is added in into system made from step 3), after 33 DEG C of fermentation 25h fermentations,
15min is centrifuged under the conditions of 10000r/min, filtrate is surpassed with the hollow-fibre membrane that retention relative molecular mass is 3KD after separation
Filter, obtained filter vacuum drying；
5) then step 4) desciccate plus 3 times of water dissolution are carried out using DA-201C type macroporous resin column chromatographies,
After being eluted with water, then with ethanol elution, de- liquid is collected, is then concentrated under reduced pressure, it is dry to get chicken embryo peptide.
The small peptide of preparation is measured using HPLC, can be obtained, chicken embryo peptide prepared by the application, relative molecular mass
Small-molecular peptides 92.3% less than 2KD.
Embodiment 2
A kind of production method of three enzymes, one ferment chicken embryo peptide, includes the following steps：
1) chicken embryo hatched 12 days is taken out, is crushed, after the water homogenate for adding 1.5 times of amounts, pH to 7 is adjusted, adds in chicken embryo quality
2% ptyalin carries out enzyme digestion reaction 1.5h under 37.5 DEG C of heat-retaining conditions；
2) and then pH to 2 is adjusted, adds in the pepsin of chicken embryo quality 2%, digested under 37.5 DEG C of heat-retaining conditions
React 3.5h；
3) and then pH to 8 is adjusted, adds in the trypsase of chicken embryo quality 1.5%, enzyme is carried out under 37.5 DEG C of heat-retaining conditions
Solution reaction 4h；
4) streptomycete of chicken embryo quality 0.2% is added in into system made from step 3), after 35 DEG C of fermentation 22h fermentations,
20min is centrifuged under the conditions of 8000r/min, filtrate is surpassed with the hollow-fibre membrane that retention relative molecular mass is 3KD after separation
Filter, obtained filter vacuum drying；
5) then step 4) desciccate plus 4 times of water dissolution are carried out using DA-201C type macroporous resin column chromatographies,
After being eluted with water, then with ethanol elution, de- liquid is collected, is then concentrated under reduced pressure, it is dry to get chicken embryo peptide.
The small peptide of preparation is measured using HPLC, can be obtained, chicken embryo peptide prepared by the application, relative molecular mass
Small-molecular peptides 91.8% less than 2KD.
Claims (10)
1. the production method of three enzymes, one ferment chicken embryo peptide, which is characterized in that the production method includes the following steps：
1) chicken embryo hatched 8-15 days is taken out, is crushed, after water is added to be homogenized, pH to 6.5-7.5 is adjusted, adds in ptyalin, in
37-37.5 enzyme digestion reaction is carried out under DEG C heat-retaining condition；
2) and then pH to 1.5-2.0 is adjusted, adds in pepsin, enzyme digestion reaction is carried out under 37-42 DEG C of heat-retaining condition；
3) and then pH to 7.5-8.0 is adjusted, adds in trypsase, enzyme digestion reaction is carried out under 37-42 DEG C of heat-retaining condition；
4) streptomycete is added in into system made from step 3), after fermentation, is centrifuged, filtrate ultrafiltration after separation, obtained filtrate is true
Sky is dry；
5) step 4) desciccate is dissolved in water, and then carries out column chromatography, is then concentrated under reduced pressure, dry to get chicken embryo peptide.
2. production method according to claim 1, which is characterized in that described in step 1) plus water homogenate refers to add in chicken embryo
The water of 1-3 times of quality.
3. production method according to claim 1 or 2, which is characterized in that the addition of ptyalin is in step 1)
The 0.6-5.5% of chicken embryo quality.
4. production method according to claim 1 or 2, which is characterized in that enzyme digestion reaction described in step 1) carries out 1-2h；
The addition of pepsin is the 0.3-3.5% of chicken embryo quality in step 2).
5. production method according to claim 1 or 2, which is characterized in that enzyme digestion reaction described in step 2) carries out 2-4h.
6. production method according to claim 1 or 5, which is characterized in that the addition of trypsase is chicken in step 3)
The 0.3-3% of idioplasm amount；Enzyme digestion reaction described in step 3) carries out 3-5h.
7. production method according to claim 1, which is characterized in that the addition of step 4) Streptomyces is chicken embryo quality
0.05-1%.
8. production method according to claim 1, which is characterized in that fermentation condition is 30-37 DEG C of fermentation 20- in step 4)
30h。
9. production method according to claim 1, which is characterized in that be dissolved in water described in step 5) and refer to add 3-5 times
Water dissolution.
10. the production method according to claim 1 or 9, which is characterized in that column chromatography described in step 5) refers to profit
With DA-201C type macroporous resin column chromatographies, after being eluted with water, then with ethanol elution, de- liquid is collected.
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| CN201810167938.0A CN108251482B (en) | 2018-02-28 | 2018-02-28 | Production method of three-enzyme one-fermentation chick embryo peptide |
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| CN201810167938.0A CN108251482B (en) | 2018-02-28 | 2018-02-28 | Production method of three-enzyme one-fermentation chick embryo peptide |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111297786A (en) * | 2020-03-20 | 2020-06-19 | 扬州扬大联环药业基因工程有限公司 | Method for extracting active small molecules from sheep embryos in large scale |
| CN114350735A (en) * | 2022-01-28 | 2022-04-15 | 贵州中医药大学第一附属医院 | Method for extracting small molecular polypeptide from chick embryo |
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| EP0688226A1 (en) * | 1992-12-17 | 1995-12-27 | PFIZER ITALIANA S.p.A. | Protein preparation for the prevention and therapy of periodontitis or other bacterial pathologies of the oral cavity |
| CN102823715A (en) * | 2012-09-19 | 2012-12-19 | 昆明理工大学 | Method for preparing bioactive peptide through full-bionic digestion model method |
| CN102871146A (en) * | 2012-10-22 | 2013-01-16 | 江南大学 | Production process of digestible chick-embryo egg health product |
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| CN102871146A (en) * | 2012-10-22 | 2013-01-16 | 江南大学 | Production process of digestible chick-embryo egg health product |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111297786A (en) * | 2020-03-20 | 2020-06-19 | 扬州扬大联环药业基因工程有限公司 | Method for extracting active small molecules from sheep embryos in large scale |
| CN114350735A (en) * | 2022-01-28 | 2022-04-15 | 贵州中医药大学第一附属医院 | Method for extracting small molecular polypeptide from chick embryo |
| CN114350735B (en) * | 2022-01-28 | 2023-08-15 | 贵州中医药大学第一附属医院 | Method for extracting small molecular polypeptides from chick embryo |
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