CN105907825A - Preparation method of casein hydrolysate peptides - Google Patents
Preparation method of casein hydrolysate peptides Download PDFInfo
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- CN105907825A CN105907825A CN201610395411.4A CN201610395411A CN105907825A CN 105907825 A CN105907825 A CN 105907825A CN 201610395411 A CN201610395411 A CN 201610395411A CN 105907825 A CN105907825 A CN 105907825A
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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
- C07K14/435—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
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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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Abstract
The invention discloses a preparation method of casein hydrolysate peptides. The preparation method comprises the following steps that 1, water is added into casein for dissolving, a casein solution with the mass concentration of 5-15wt% is prepared, and pH is adjusted to be 6-9; 2, trypsin and an enzyme vigor-sustaining agent are added into the casein solution, hydrolysis is carried out for 1.5-4.5 h under the temperature of 45-75 DEG C, enzyme activity is killed, and enzymatic hydrolysate is obtained; 3, the pH value of the enzymatic hydrolysate is adjusted to be 4-5 for isoelectric precipitation, centrifugal separation is carried out to obtain supernate, the supernate and antioxidant are mixed according to the mass ratio of (95-105):(0.05-0.5) to be uniform to obtain a mixed solution; 4, ultrafiltration and freeze-drying are carried out. According to the preparation method of casein hydrolysate peptides, the degree of hydrolysis is moderate, casein hydrolysate peptides can be easily absorbed in the body, the preparation process is simple, environmentally friendly and free of pollution, the production cost is greatly reduced, and the prepared product can enhance immunity, resists aging and has the good application value.
Description
Technical field
The present invention relates to technical field of health care food, be specifically related to the preparation method of a kind of casein hydrolysate peptides.
Background technology
Casein contains eight kinds of aminoacid of needed by human, is that (PER value is a kind of complete protein
2.5), it is possible to provide essential amino acid for organism growth promoter.Casein is a kind of phosphorous combination
Albumen, with the hydroxy combining of phosphoric acid ester bond Yu serine, in addition with a small amount of hexose, amino sugar and saliva
The residues such as acid, its outstanding feature is at 20 DEG C, can precipitate under the conditions of pH4.6.
Along with the raising day by day of quality of the life, lactoprotein is as one of mankind's high-quality protein source, again always
Concerned.By the peptide chain of macro-molecular protein being hydrolyzed into the peptide fragment of little molecule, its battalion can not only be improved
Support and be worth, improve physicochemical property, more can obtain the Functional Polypeptides with multiple physiologically active.Small-molecular peptides exists
Internal digesting and assimilating fast, protein utilization is high；There is low antigenicity, anaphylaxis will not be produced；Tool
There are the excellent physical properties such as highly dissoluble, low viscosity, high fluidity and heat stability；Generate one simultaneously
Series has the peptide fragment of sp act, if any calmness, the hydrolyzed casein of sedative action, can suppress vasotonia
Transin enzyme (ACE) activity blood pressure lowering peptide, have function vector mineral element absorption promote peptide,
Resist antibacterial and the antibacterial peptide etc. of virus infection.
The preparation method of biologically active peptide mainly has three kinds at present: (1) extraction method, i.e. from the life of nature
Extracting itself intrinsic natural activity peptide in object, it is big that it extracts difficulty, and raw material sources are limited, product
Cost is high；(2) Hydrolyze method, will be hydrolyzed to obtain the biological activity with physiological function by protein
Peptide；(3) synthetic method, the i.e. method of applied chemistry synthesis prepare biologically active peptide, but reagent, instrument
Relatively costly, side reaction is many, and there is residual compounds, and application is restricted.Based on different purposes
Selecting different preparation methoies, different preparation methoies respectively has pluses and minuses.
The invention provides the preparation method of a kind of casein hydrolysate peptides, it is easy to absorb, and can effectively drop
The content of anaphylactogen in low casein.
Summary of the invention
For the deficiencies in the prior art, the technical problem to be solved is to provide a kind of casein water
Solve the preparation method of peptide.
For achieving the above object, the adopted technical solution is that:
The preparation method of a kind of casein hydrolysate peptides, comprises the following steps:
(1) casein is dissolved in water it is configured to the casein solution that mass concentration is 5-15wt%, and adjust
Joint pH to 5-10；
(2) trypsin and enzyme live keeping agent are added in casein solution, at 45-75 DEG C, hydrolyze 1.5-4.5
Hour, enzyme denaturing is lived, and obtains enzymolysis solution；
(3) pH value of regulation enzymolysis solution is to 4-5 isoelectric precipitation, centrifugation, obtains supernatant,
By supernatant and antioxidant in mass ratio for (95-105): (0.05-0.5) is mixed homogeneously and obtained mixed liquor；
(4) ultrafiltration, lyophilizing.
In described step (1) and step (3), regulation pH value can use NaOH solution, HCl
Solution etc..
Preferably, the 0.5-5% that addition is casein solution quality of enzyme, enzyme in described step (2)
The addition of live keeping agent is the 0.1-1% of casein solution quality.
Preferably, the enzyme live keeping agent in described step (2) is by mannitol, hydroxyl isomaltulose, trehalose
Mixing, described mannitol, hydroxyl isomaltulose, the mass ratio of trehalose are (1-3): (1-3):
(1-3)。
Preferably, the antioxidant in described step (3) is mixed by licochalcone A, morin, 4',5,7-trihydroxyflavone
Conjunction forms, and described licochalcone A, morin, the mass ratio of 4',5,7-trihydroxyflavone are (1-3): (1-3):
(1-3)。
Preferably, the centrifugation rotating speed in described step (3) is 2500-4500 rev/min, and the time is
25-35 minute.
Preferably, the ultrafiltration in described step (4) is with the ultrafilter membrane ultrafiltration of molecular cut off 1-10kD.
The preparation method of casein hydrolysate peptides of the present invention, degree of hydrolysis is moderate, it is easy to body absorption, prepares work
Skill is simple, environment friendly and pollution-free, greatly reduces production cost, the product of preparation can enhancing immunity,
Defying age, has preferable using value.
Detailed description of the invention
Each raw material introduction in embodiment:
Casein, Casein, No. CAS: 9000-71-9.Use Song Guan bio tech ltd, Nanjing
The food grade caseins produced.
Mannitol, No. CAS: 69-65-8.
Hydroxyl isomaltulose, No. CAS: 64519-82-0.
Trehalose, No. CAS: 99-20-7.
Trypsin, No. CAS: 9002-07-7, Zhengzhou good brother chemical products company limited provides, enzyme
Live 200U/mg.
Licochalcone A, No. CAS: 58749-22-7.
Morin, No. CAS: 480-16-0.
4',5,7-trihydroxyflavone, No. CAS: 520-36-5.
Embodiment 1
The preparation method of casein hydrolysate peptides, comprises the following steps:
(1) casein deionized water dissolving is configured to the casein solution that mass concentration is 10wt%
(the most every 10 grams of caseins of 100 grams of deionized water dissolving), and adjust with the sodium hydroxide solution of 2mol/L
Joint pH to 8.0；
(2) will trypsin and enzyme live keeping agent addition casein solution be uniformly mixed, wherein pancreas egg
Addition is casein solution quality the 1.2% of white enzyme, the addition of enzyme live keeping agent is casein solution matter
0.6% (the most every 100 grams of casein solution add trypsin 1.2 grams, enzyme live keeping agent 0.6 gram) of amount,
Hydrolyze 2 hours at 50 DEG C, obtain hydrolyzing afterproduct, then hydrolysis afterproduct is placed under 95 DEG C of water-baths
Within 10 minutes, enzyme denaturing is lived, and obtains enzymolysis solution；
(3) with the pH value of the hydrochloric acid solution regulation enzymolysis solution of 2mol/L to 4.6 casein isoelectric precipitations,
After precipitation to be precipitated, with 3000r/min centrifugation 30min remove unhydrolysed casein, obtain from
Supernatant after the heart, mixs homogeneously supernatant for 100:0.3 in mass ratio with antioxidant and obtains mixed liquor；
(4) by the mixed liquor ultrafilter membrane ultrafiltration of molecular cut off 6kD, filtrate is collected, at-30 DEG C
Lyophilizing, obtains the casein hydrolysate peptides of embodiment 1 preparation.
Enzyme live keeping agent in described step (2) is by mannitol, hydroxyl isomaltulose, trehalose in mass ratio
It is uniformly mixed for 1:1:1 and obtains.
Antioxidant in described step (3) is mixed by licochalcone A, morin, 4',5,7-trihydroxyflavone,
Described licochalcone A, morin, the mass ratio of 4',5,7-trihydroxyflavone are 1:1:1.
Embodiment 2
Substantially the same manner as Example 1, differ only in: described enzyme live keeping agent is by mannitol, different wheat
Bud ketose alcohol is uniformly mixed for 1:1 in mass ratio and obtains.Can be prepared by the casein water of embodiment 2
Solve peptide.
Embodiment 3
Substantially the same manner as Example 1, differ only in: described enzyme live keeping agent is by mannitol, Sargassum
Sugar is uniformly mixed for 1:1 in mass ratio and obtains.Can be prepared by the casein hydrolysate peptides of embodiment 3.
Embodiment 4
Substantially the same manner as Example 1, differ only in: described enzyme live keeping agent by hydroxyl isomaltulose,
Trehalose is uniformly mixed for 1:1 in mass ratio and obtains.Can be prepared by the casein hydrolysis of embodiment 4
Peptide.
Embodiment 5
Substantially the same manner as Example 1, differ only in: described antioxidant by licochalcone A,
Morin is uniformly mixed for 1:1 in mass ratio and obtains.Can be prepared by the casein hydrolysis of embodiment 5
Peptide.
Embodiment 6
Substantially the same manner as Example 1, differ only in: described antioxidant by licochalcone A,
4',5,7-trihydroxyflavone is uniformly mixed for 1:1 in mass ratio and obtains.Can be prepared by the casein hydrolysis of embodiment 6
Peptide.
Embodiment 7
Substantially the same manner as Example 1, differ only in: described antioxidant is by morin, 4',5,7-trihydroxyflavone
It is uniformly mixed for 1:1 in mass ratio and obtains.Can be prepared by the casein hydrolysate peptides of embodiment 7.
Test case 1
The enzymolysis solution obtaining embodiment 1-7 step (2) tests its degree of hydrolysis, and concrete outcome is shown in Table 1:
Table 1: casein hydrolysis degree test table
| Degree of hydrolysis/% | |
| Embodiment 1 | 18.32 |
| Embodiment 2 | 9.12 |
| Embodiment 3 | 9.08 |
| Embodiment 4 | 8.78. |
| Embodiment 5 | 8.69 |
| Embodiment 6 | 8.95 |
| Embodiment 7 | 7.82 |
Comparing embodiment 1 and embodiment 2-4, embodiment 1 (mannitol, hydroxyl isomaltulose, trehalose
Compounding) degree of hydrolysis is apparently higher than embodiment 2-4 (in mannitol, hydroxyl isomaltulose, trehalose arbitrarily
Two kinds compound).Comparing embodiment 1 and embodiment 5-7, embodiment 1 (licochalcone A, morin,
4',5,7-trihydroxyflavone compounds) degree of hydrolysis is apparently higher than embodiment 2-4 (licochalcone A, morin, 4',5,7-trihydroxyflavone
In any two kinds compound).
Test case 2
Enzymolysis solution prepared by casein solution embodiment 1-7 step (1) prepared and step (2) enters
Row allergen content is tested, and calculates anaphylactogen reduction rate, and computing formula is: anaphylactogen reduction rate (%)
In=(allergen content in allergen content-enzymolysis solution in casein solution)/casein solution, anaphylactogen contains
Amount × 100%, concrete outcome is shown in Table 2.
Table 2: anaphylactogen reduction rate
| Anaphylactogen reduction rate/% | |
| Embodiment 1 | 99.94 |
| Embodiment 2 | 92.65 |
| Embodiment 3 | 93.74 |
| Embodiment 4 | 93.83 |
| Embodiment 5 | 92.56 |
| Embodiment 6 | 93.62 |
| Embodiment 7 | 92.79 |
Comparing embodiment 1 and embodiment 2-4, embodiment 1 (mannitol, hydroxyl isomaltulose, trehalose
Compounding) anaphylactogen reduction rate is apparently higher than embodiment 2-4 (mannitol, hydroxyl isomaltulose, trehalose
In any two kinds compound).Comparing embodiment 1 and embodiment 5-7, embodiment 1 (licochalcone A,
Morin, 4',5,7-trihydroxyflavone are compounding) anaphylactogen reduction rate apparently higher than embodiment 2-4 (licochalcone A,
During morin, 4',5,7-trihydroxyflavone are compounding, any two kinds compound).
Test case 3
Casein hydrolysate peptides embodiment 1-7 prepared, is placed in 25 DEG C, protects under relative humidity 85% environment
Hide half a year, use " GB/T 4789.2-2010 microbiological test of food hygiene total plate count mensuration "
Carry out total plate count test.Concrete test result is shown in Table 3.
Table 3: total plate count test table cfu/g
| Total plate count | |
| Embodiment 1 | 1.8×103 |
| Embodiment 2 | 4.3×103 |
| Embodiment 3 | 3.6×103 |
| Embodiment 4 | 4.2×103 |
| Embodiment 5 | 4.8×103 |
| Embodiment 6 | 4.9×103 |
| Embodiment 7 | 4.5×103 |
Inventor is found surprisingly that, comparing embodiment 1 and embodiment 2-4, and embodiment 1 is (mannitol, different
Maltulose alcohol, trehalose are compounding) anti-microbial property is apparently higher than embodiment 2-4 (mannitol, different Fructus Hordei Germinatus
In ketose alcohol, trehalose, any two kinds compound).Comparing embodiment 1 and embodiment 5-7, embodiment 1
(licochalcone A, morin, 4',5,7-trihydroxyflavone are compounding) anti-microbial property is (sweet apparently higher than embodiment 2-4
During grass chalcone A, morin, 4',5,7-trihydroxyflavone are compounding, any two kinds compound).
Claims (4)
1. the preparation method of a casein hydrolysate peptides, it is characterised in that comprise the following steps:
(1) casein is dissolved in water into the casein solution of 5-15wt%, and regulates pH to 6-9；
(2) trypsin and enzyme live keeping agent are added in casein solution, at 45-75 DEG C, hydrolyze 1.5-4.5
Hour, enzyme denaturing is lived, and obtains enzymolysis solution；
(3) pH value of regulation enzymolysis solution is to 4-5, isoelectric precipitation, centrifugation, obtains supernatant,
By supernatant and antioxidant in mass ratio for (95-105): (0.05-0.5) is mixed homogeneously and obtained mixed liquor；
(4) ultrafiltration, lyophilizing；
Described antioxidant is mixed by licochalcone A, morin, 4',5,7-trihydroxyflavone, and described Radix Glycyrrhizae is looked into
That ketone A, morin, the mass ratio of 4',5,7-trihydroxyflavone are (1-3): (1-3): (1-3).
Described enzyme live keeping agent is mixed by mannitol, hydroxyl isomaltulose, trehalose, described manna
Alcohol, hydroxyl isomaltulose, the mass ratio of trehalose are (1-3): (1-3): (1-3).
2. the preparation method of casein hydrolysate peptides as claimed in claim 1, it is characterised in that: described
Centrifugation rotating speed is 2500-4500 rev/min, and the time is 25-35 minute.
3. the preparation method of casein hydrolysate peptides as claimed in claim 1, it is characterised in that: described
Ultrafiltration is with the ultrafilter membrane ultrafiltration of molecular cut off 1-10kD.
4. the preparation method of casein hydrolysate peptides as claimed in claim 1, it is characterised in that: described step
Suddenly the 0.5-5% that addition is casein solution quality of enzyme in (2), the addition of enzyme live keeping agent is cheese
The 0.1-1% of protein solution quality.
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| CN201610395411.4A CN105907825A (en) | 2016-06-06 | 2016-06-06 | Preparation method of casein hydrolysate peptides |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107460037A (en) * | 2017-09-30 | 2017-12-12 | 陈芹芳 | High calcium camellia oil and its production technology |
| CN108467487A (en) * | 2018-03-30 | 2018-08-31 | 合肥工业大学 | The dextrin modified casein polypeptide conjugate of starch base, preparation method and application |
| CN109431950A (en) * | 2018-11-12 | 2019-03-08 | 宁波蒙曼生物科技有限公司 | A kind of anti-wrinkle face mask liquid and preparation method thereof |
-
2016
- 2016-06-06 CN CN201610395411.4A patent/CN105907825A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107460037A (en) * | 2017-09-30 | 2017-12-12 | 陈芹芳 | High calcium camellia oil and its production technology |
| CN108467487A (en) * | 2018-03-30 | 2018-08-31 | 合肥工业大学 | The dextrin modified casein polypeptide conjugate of starch base, preparation method and application |
| CN109431950A (en) * | 2018-11-12 | 2019-03-08 | 宁波蒙曼生物科技有限公司 | A kind of anti-wrinkle face mask liquid and preparation method thereof |
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Application publication date: 20160831 |