CN108866136A - A kind of preparation method of low molecular polypeptide - Google Patents
A kind of preparation method of low molecular polypeptide Download PDFInfo
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- CN108866136A CN108866136A CN201810867788.4A CN201810867788A CN108866136A CN 108866136 A CN108866136 A CN 108866136A CN 201810867788 A CN201810867788 A CN 201810867788A CN 108866136 A CN108866136 A CN 108866136A
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- diatomite
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- sodium chloride
- ethyl alcohol
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 22
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 20
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 65
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 60
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 39
- 235000019441 ethanol Nutrition 0.000 claims abstract description 35
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000011780 sodium chloride Substances 0.000 claims abstract description 30
- 239000007788 liquid Substances 0.000 claims abstract description 29
- 239000002002 slurry Substances 0.000 claims abstract description 22
- 210000002826 placenta Anatomy 0.000 claims abstract description 18
- 239000000287 crude extract Substances 0.000 claims abstract description 17
- 238000003825 pressing Methods 0.000 claims abstract description 16
- 239000000706 filtrate Substances 0.000 claims abstract description 15
- 238000003756 stirring Methods 0.000 claims abstract description 15
- 235000015099 wheat brans Nutrition 0.000 claims abstract description 15
- 241000283707 Capra Species 0.000 claims abstract description 12
- 230000007071 enzymatic hydrolysis Effects 0.000 claims abstract description 10
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims abstract description 10
- 230000001376 precipitating effect Effects 0.000 claims abstract description 9
- 239000000284 extract Substances 0.000 claims abstract description 8
- 238000002203 pretreatment Methods 0.000 claims abstract description 8
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 238000010792 warming Methods 0.000 claims abstract description 8
- 238000007710 freezing Methods 0.000 claims abstract description 4
- 230000008014 freezing Effects 0.000 claims abstract description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 28
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 14
- 230000000903 blocking effect Effects 0.000 claims description 7
- 210000004204 blood vessel Anatomy 0.000 claims description 7
- 210000003195 fascia Anatomy 0.000 claims description 7
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 5
- 238000006243 chemical reaction Methods 0.000 abstract 1
- 230000008030 elimination Effects 0.000 abstract 1
- 238000003379 elimination reaction Methods 0.000 abstract 1
- 238000001914 filtration Methods 0.000 abstract 1
- 239000012535 impurity Substances 0.000 abstract 1
- 239000008188 pellet Substances 0.000 abstract 1
- 238000010298 pulverizing process Methods 0.000 abstract 1
- 238000010008 shearing Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 14
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 9
- 239000000523 sample Substances 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- -1 hydroxyl radical free radical Chemical class 0.000 description 3
- 230000003078 antioxidant effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- XNOPRXBHLZRZKH-DSZYJQQASA-N oxytocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 XNOPRXBHLZRZKH-DSZYJQQASA-N 0.000 description 2
- OCZVHBZNPVABKX-UHFFFAOYSA-N 1,1-diphenyl-2-(2,4,6-trinitrophenyl)hydrazine;ethanol Chemical compound CCO.[O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1NN(C=1C=CC=CC=1)C1=CC=CC=C1 OCZVHBZNPVABKX-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 102400000050 Oxytocin Human genes 0.000 description 1
- XNOPRXBHLZRZKH-UHFFFAOYSA-N Oxytocin Natural products N1C(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CC(C)C)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C(C(C)CC)NC(=O)C1CC1=CC=C(O)C=C1 XNOPRXBHLZRZKH-UHFFFAOYSA-N 0.000 description 1
- 101800000989 Oxytocin Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 150000002831 nitrogen free-radicals Chemical class 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000001151 other effect Effects 0.000 description 1
- 229960001723 oxytocin Drugs 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 125000002467 phosphate group Chemical class [H]OP(=O)(O[H])O[*] 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000037384 skin absorption Effects 0.000 description 1
- 231100000274 skin absorption Toxicity 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 235000015961 tonic Nutrition 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 229960000716 tonics Drugs 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
Classifications
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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
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses a kind of preparation method of low molecular polypeptide, which includes the following steps：（1）Pre-treatment：Goat Placenta impurity elimination is washed, is dried, shearing, freezing, pulverizing；（2）Enzymatic hydrolysis：Into slurries plus water, tune pH to 7.7 ~ 7.8 add trypsase and Protamex, are warming up to 40 ~ 43 DEG C of reactions, take filtrate after filter paper filtering；（3）It extracts：Into enzymolysis liquid plus ethyl alcohol, sodium chloride and diatomite, tune pH to 6.4 ~ 6.6 stir rear filters pressing at 10 ~ 15 DEG C, take supernatant；Into crude extract plus ethyl alcohol, sodium chloride, diatomite and wheat bran, tune pH to 6.8 ~ 7.0 stir rear filters pressing at 3 ~ 5 DEG C, take precipitating；Take pellet frozen, pulverized at 0 ~ 10 DEG C, add water, rear organic filter film is stirred at 10 ~ 15 DEG C and filters and take filtrate, freezing to get；In the process when temperature control, control amount, control etc., low molecular polypeptide is obtained.
Description
Technical field
The present invention relates to a kind of preparation methods of low molecular polypeptide.
Background technique
Placenta, which is rich in, breeds all primitive life substances necessary to life.Currently, placenta especially Goat Placenta is medicinal
It is had been to be concerned by more and more people with health-care efficacy.For Goat Placenta, wherein protein rich in, 17 kinds of amino acid,
14 kinds of microelements, phosphatide, lipopolysaccharides, vitamins, there are also more to immune function and the relevant various active of body normal operation
Peptide, demand of the ratio that nutritional ingredient is naturally arranged in pairs or groups closest to human body.《Compendium of Materia Medica》In i.e. to Goat Placenta the effect of
It records：" sheep placenta, the tire beast in ewe abdomen is sweet in flavor and warm in property, have QI invigorating qi-restoratives, middle benefit gas it is warm under, adjust that kidney tonifying is empty, thin thin function
Effect ".Scientist confirms that it is big that Goat Placenta, which also has nourishing blood and tranquilization, riching and moisturizing skim, promotes longevity and other effects, by many experiments
The strengthening by means of tonics medicine of tonifying primordial Qi.In conclusion the functional characteristic of placenta is increasingly recognized by people, placenta objectively also proposed
The requirement of deep processing.Due to containing a large amount of active materials needed by human in placenta, therefrom the polypeptide of extraction functionality has
There is wide development prospect.
However contain comparable high molecular weight polypeptide in the polypeptide prepared in the prior art, it is unfavorable for skin absorption, limitation
Its application.
Summary of the invention
The object of the present invention is to provide a kind of preparation methods of low molecular polypeptide, to obtain low molecular polypeptide.
Above-mentioned technical purpose of the invention technical scheme is that：
A kind of preparation method of low molecular polypeptide, includes the following steps：
(1) pre-treatment
Fresh Goat Placenta is taken, removes fascia and blood vessel, washing, dry in the air 1~2hr at room temperature；Shear it is blocking, in -30~-20
8~12hr is freezed at DEG C, is pulverized at 0~10 DEG C and is obtained slurries；
(2) it digests
Under stirring condition, water is added into slurries, the aqueous sodium carbonate regulation system pH of 0.8~1.1mol/l is added extremely
7.7~7.8, trypsase and Protamex is added, is warming up to 40~43 DEG C and is reacted, until enzymatic hydrolysis is completely, is filtered with filter paper
After take filtrate, as enzymolysis liquid；
By mass, the amount ratio of slurries, water, trypsase and Protamex be 1: 4~6: 0.2~0.4: 0.1~
0.2；
(3) it extracts
Ethyl alcohol, sodium chloride and diatomite are added into enzymolysis liquid, the aqueous hydrochloric acid solution that 0.8~1.1mol/l is added adjusts body
It is pH to 6.4~6.6,30~45min is stirred at 10~15 DEG C, with 1.6~2.0Kg/cm at 10~15 DEG C2Pressure into
Row filters pressing takes supernatant, as crude extract；By mass, the amount ratio of enzymolysis liquid, ethyl alcohol, sodium chloride and diatomite is 1:
0.08~0.12: 0.04~0.06: 0.05~0.08；
Ethyl alcohol, sodium chloride, diatomite and wheat bran are added into crude extract, the aqueous hydrochloric acid solution of 0.8~1.1mol/1 is added
Regulation system pH to 6.8~7.0 stirs 30~45min, with 1.6~2.0Kg/cm at 3~5 DEG C at 3~5 DEG C2Pressure
Filters pressing is carried out, precipitating is taken；By mass, the amount ratio of crude extract, ethyl alcohol, sodium chloride, diatomite and wheat bran be 1: 0.08~
0.12: 0.02~0.04: 0.03~0.05: 0.04~0.06；
It takes and is deposited in 8~12hr of freezing at -30~-20 DEG C, pulverized at 0~10 DEG C, water is added, at 10~15 DEG C
30~45min of lower stirring filters and takes filtrate with 0.45 μm or 0.22 μm of organic filter film, at -30~-20 DEG C freeze 8~
12hr to get；By mass, it precipitates and the amount ratio of water is 1: 2~3.
Further preferably：Include the following steps：
(1) pre-treatment
Fresh Goat Placenta is taken, removes fascia and blood vessel, washing, dry in the air 1.5hr at room temperature；Shear it is blocking, it is cold at -25 DEG C
Freeze 10hr, is pulverized at 4 DEG C and obtain slurries；
(2) it digests
Under stirring condition, water is added into slurries, the aqueous sodium carbonate regulation system pH to 7.8 of 1.0mol/l is added,
Trypsase and Protamex is added, is warming up to 42 DEG C and is reacted, until enzymatic hydrolysis is completely, filtrate is taken after being filtered with filter paper, as
Enzymolysis liquid；
By mass, the amount ratio of slurries, water, trypsase and Protamex are 1: 5: 0.3: 0.15；
(3) it extracts
Ethyl alcohol, sodium chloride and diatomite are added into enzymolysis liquid, the aqueous hydrochloric acid solution regulation system pH of 1.0mol/l is added
To 6.5,40min is stirred at 12 DEG C, with 1.8Kg/cm at 12 DEG C2Pressure carry out filters pressing, take supernatant, as slightly mention
Liquid；By mass, the amount ratio of enzymolysis liquid, ethyl alcohol, sodium chloride and diatomite is 1: 0.10: 0.05: 0.06；
Ethyl alcohol, sodium chloride, diatomite and wheat bran are added into crude extract, the aqueous hydrochloric acid solution that 1.0mol/l is added adjusts body
It is pH to 6.9, stirs 40min at 4 DEG C, with 1.8Kg/cm at 4 DEG C2Pressure carry out filters pressing, take precipitating；By mass,
Crude extract, ethyl alcohol, sodium chloride, diatomite and wheat bran amount ratio be 1: 0.10: 0.03: 0.04: 0.05；
It takes to be deposited at -25 DEG C and freezes 10hr, pulverized at 4 DEG C, water is added, 40min is stirred at 12 DEG C, use
0.22 μm of organic filter film filters and takes filtrate, at -25 DEG C freeze 10hr to get；By mass, the amount ratio with water is precipitated
It is 1: 2.4.
Further preferably：The second alcohol and water added in every step of step (3) passes through the pre-heat treatment, the temperature of preheating with
The temperature that it is stirred to react is consistent.
In conclusion the invention has the advantages that：Low molecular polypeptide is obtained by the method for chemical-biological, it is low
Molecular polypeptide content is higher, is conducive to absorb, and improves its absorption and effect in skin care item；Film is applied compared to the prior art
Or the methods of chromatography obtains to separate, the present processes cost is lower, reduces absorption of the substance on film or chromatography etc. and causes
A possibility that equipment needs to be replaced frequently.
Specific embodiment
This specific embodiment is only explanation of the invention, is not limitation of the present invention, those skilled in the art
Member can according to need the modification that not creative contribution is made to the present embodiment after reading this specification, but as long as at this
All by the protection of Patent Law in the protection scope of invention.
Embodiment 1：A kind of preparation method of low molecular polypeptide, includes the following steps：
(1) pre-treatment
Fresh Goat Placenta is taken, removes fascia and blood vessel, washing, dry in the air 1.5hr at room temperature；Shear it is blocking, it is cold at -25 DEG C
Freeze 10hr, is pulverized at 4 DEG C and obtain slurries；
(2) it digests
Under stirring condition, water is added into slurries, the aqueous sodium carbonate regulation system pH to 7.8 of 1.0mol/1 is added,
Trypsase and Protamex is added, is warming up to 42 DEG C and is reacted, until enzymatic hydrolysis is completely, filtrate is taken after being filtered with filter paper, as
Enzymolysis liquid；
By mass, the amount ratio of slurries, water, trypsase and Protamex are 1: 5: 0.3: 0.15；
(3) it extracts
Ethyl alcohol, sodium chloride and diatomite are added into enzymolysis liquid, the aqueous hydrochloric acid solution regulation system pH of 1.0mol/1 is added
To 6.5,40min is stirred at 12 DEG C, with 1.8Kg/cm at 12 DEG C2Pressure carry out filters pressing, take supernatant, as slightly mention
Liquid；By mass, the amount ratio of enzymolysis liquid, ethyl alcohol, sodium chloride and diatomite is 1: 0.10: 0.05: 0.06；
Ethyl alcohol, sodium chloride, diatomite and wheat bran are added into crude extract, the aqueous hydrochloric acid solution that 1.0mol/l is added adjusts body
It is pH to 6.9, stirs 40min at 4 DEG C, with 1.8Kg/cm at 4 DEG C2Pressure carry out filters pressing, take precipitating；By mass,
Crude extract, ethyl alcohol, sodium chloride, diatomite and wheat bran amount ratio be 1: 0.10: 0.03: 0.04: 0.05；
It takes to be deposited at -25 DEG C and freezes 10hr, pulverized at 4 DEG C, water is added, 40min is stirred at 12 DEG C, use
0.22 μm of organic filter film filters and takes filtrate, at -25 DEG C freeze 10hr to get；By mass, the amount ratio with water is precipitated
It is 1: 2.4；
The second alcohol and water added in every step of step (3) passes through the pre-heat treatment, and the temperature of preheating is stirred to react with it
Temperature is consistent.
Embodiment 2：A kind of preparation method of low molecular polypeptide, includes the following steps：
(1) pre-treatment
Fresh Goat Placenta is taken, removes fascia and blood vessel, washing, dry in the air 1hr at room temperature；It shears blocking, is freezed at -30 DEG C
12hr is pulverized at 0 DEG C and is obtained slurries；
(2) it digests
Under stirring condition, water is added into slurries, the aqueous sodium carbonate regulation system pH to 7.7 of 0.8mol/l is added,
Trypsase and Protamex is added, is warming up to 40 DEG C and is reacted, until enzymatic hydrolysis is completely, filtrate is taken after being filtered with filter paper, as
Enzymolysis liquid；
By mass, the amount ratio of slurries, water, trypsase and Protamex are 1: 4: 0.2: 0.1；
(3) it extracts
Ethyl alcohol, sodium chloride and diatomite are added into enzymolysis liquid, the aqueous hydrochloric acid solution regulation system pH of 0.8mol/l is added
To 6.4,45min is stirred at 10 DEG C, with 2.0Kg/cm at 10 DEG C2Pressure carry out filters pressing, take supernatant, as slightly mention
Liquid；By mass, the amount ratio of enzymolysis liquid, ethyl alcohol, sodium chloride and diatomite is 1: 0.08: 0.04: 0.05；
Ethyl alcohol, sodium chloride, diatomite and wheat bran are added into crude extract, the aqueous hydrochloric acid solution that 0.8mol/l is added adjusts body
It is pH to 6.8, stirs 45min at 3 DEG C, with 2.0Kg/cm at 3 DEG C2Pressure carry out filters pressing, take precipitating；By mass,
Crude extract, ethyl alcohol, sodium chloride, diatomite and wheat bran amount ratio be 1: 0.08: 0.02: 0.03: 0.04；
It takes to be deposited at -30 DEG C and freezes 12hr, pulverized at 0 DEG C, water is added, 45min is stirred at 10 DEG C, use
0.45 μm of organic filter film filters and takes filtrate, at -30 DEG C freeze 12hr to get；By mass, the amount ratio with water is precipitated
It is 1: 2；
The second alcohol and water added in every step of step (3) passes through the pre-heat treatment, and the temperature of preheating is stirred to react with it
Temperature is consistent.
Embodiment 3：A kind of preparation method of low molecular polypeptide, includes the following steps：
(1) pre-treatment
Fresh Goat Placenta is taken, removes fascia and blood vessel, washing, dry in the air 2hr at room temperature；It shears blocking, is freezed at -20 DEG C
8hr is pulverized at 10 DEG C and is obtained slurries；
(2) it digests
Under stirring condition, water is added into slurries, the aqueous sodium carbonate regulation system pH to 7.8 of 1.1mol/l is added,
Trypsase and Protamex is added, is warming up to 43 DEG C and is reacted, until enzymatic hydrolysis is completely, filtrate is taken after being filtered with filter paper, as
Enzymolysis liquid；
By mass, the amount ratio of slurries, water, trypsase and Protamex are 1: 6: 0.4: 0.2；
(3) it extracts
Ethyl alcohol, sodium chloride and diatomite are added into enzymolysis liquid, the aqueous hydrochloric acid solution regulation system pH of 1.1mol/l is added
To 6.6,30min is stirred at 15 DEG C, with 1.6Kg/cm at 15 DEG C2Pressure carry out filters pressing, take supernatant, as slightly mention
Liquid；By mass, the amount ratio of enzymolysis liquid, ethyl alcohol, sodium chloride and diatomite is 1: 0.12: 0.06: 0.08；
Ethyl alcohol, sodium chloride, diatomite and wheat bran are added into crude extract, the aqueous hydrochloric acid solution that 1.1mol/l is added adjusts body
It is pH to 7.0,30min is stirred at 5 DEG C, at 5 DEG C with 1.6Kg/cm2Pressure carry out filters pressing, take precipitating；By mass,
Crude extract, ethyl alcohol, sodium chloride, diatomite and wheat bran amount ratio be 1: 0.12: 0.04: 0.05: 0.06；
It takes to be deposited at -20 DEG C and freezes 8hr, pulverized at 10 DEG C, water is added, 30min is stirred at 15 DEG C, use
0.22 μm of organic filter film filters and takes filtrate, at -20 DEG C freeze 8hr to get；By mass, the amount ratio with water is precipitated
It is 1: 3；
The second alcohol and water added in every step of step (3) passes through the pre-heat treatment, and the temperature of preheating is stirred to react with it
Temperature is consistent.
Performance characterization
1, gel chromatography measures molecular weight
After the Tris-HCl eluent Balance Treatment SphedexG-25 gel column of 0.1mol/l, in wavelength X=220nm
Under conditions of, detectable concentration is the standard substance of 2mg/ml：The sweet peptide of reduced form Gu light (307Da), oxytocin (1008Da) and pancreas
The gel chromatography of glucagons (3483Da) obtains standard according to standard substance molecular weight logarithm (lgMr) and elution volume (Ve)
Curve and regression equation.Taking concentration is that the test sample of 5mg/ml is analyzed under above-mentioned similarity condition, by the appearance of sample
Volume and standard curve are compared, and obtain the molecular weight and its distribution of sample.Different points are determined with peak area normalization method
The percentage contents of son amount range polypeptide.It parallel test 3 times, is averaged.
Test result is as shown in table 1.Table 1 shows that the content of embodiment 1-3 middle-molecular-weihydroxyethyl > 5000Da is below 1.5%,
The content of molecular weight < 1000Da is above 87%, hence it is evident that is lower than the requirement of 1.5KDa to molecular weight better than cosmetics；It is wherein real
It is best for applying example 1.
1 gel chromatography of table measures molecular weight results
2, antioxidant activity in vitro measures
Measuring principle：DPPH.free radical (DPPH, C18H12N5O6) it is a kind of very stable centered on nitrogen
Free radical prompt tested material to have if tested material can remove it and remove hydroxyl radical free radical, alkane free radical or peroxy radical
The effects of.DPPH ethanol water is in navy blue, has strong absorption in 520nm, can measure its absorbance with spectrophotometer, is added
After tested material, absorbance is caused to reduce because removing DPPH.Tested material can be evaluated according to absorbance change situation and remove DPPH's
Ability.
Sample measurement：1. 6.88 standard phosphate salt buffer solution 4ml of pH value is sequentially added into 10ml test tube,
0.1777mol/l DPPH solution 4ml is mixed；2. adding testing sample solution, distilled water supplements volume to 10.00ml, fills
Divide and mixes；3. absorbance is measured after 10min at 520nm；Every group of sample is repeated three times, and is averaged；4. certainly to DPPH
By clearance rate=[1- (A1-A2)/A3] × 100% of base；A1 indicates DPPH solution absorbance after adding sample to be tested to react；A2 table
Show and DPPH is not added, only adds the solution absorbance of sample to be tested and water；A3 expression does not add sample to be tested, only adds the solution of DPPH and water
Absorbance.It parallel test 3 times, is averaged.
The results are shown in Table 2.The display of table 2 is compared with enzymolysis liquid, and embodiment 1-3 is higher to the clearance rate of DPPH free radical,
Wherein embodiment 1 is best.
2 antioxidant activity in vitro measurement result of table
The specific implementation of the invention is not to be limited to these illustrations for the above content, and technology belonging to the present invention is led
For the those of ordinary skill in domain, without departing from the inventive concept of the premise, a number of simple deductions or replacements can also be made,
It all shall be regarded as belonging to present invention scope of patent protection determined by the appended claims.
Claims (3)
1. a kind of preparation method of low molecular polypeptide, which is characterized in that include the following steps：
（1）Pre-treatment
Fresh Goat Placenta is taken, removes fascia and blood vessel, washing, dry in the air 1 ~ 2hr at room temperature；Shear it is blocking, it is cold at -30 ~ -20 DEG C
Freeze 8 ~ 12hr, is pulverized at 0 ~ 10 DEG C and obtain slurries；
（2）Enzymatic hydrolysis
Under stirring condition, be added water into slurries, be added the aqueous sodium carbonate regulation system pH to 7.7 of 0.8 ~ 1.1mol/l ~
7.8, trypsase and Protamex is added, is warming up to 40 ~ 43 DEG C and is reacted, until enzymatic hydrolysis is completely, filter is taken after being filtered with filter paper
Liquid, as enzymolysis liquid；
By mass, the amount ratio of slurries, water, trypsase and Protamex are 1:4~6:0.2~0.4:0.1~0.2；
（3）It extracts
Ethyl alcohol, sodium chloride and diatomite are added into enzymolysis liquid, the aqueous hydrochloric acid solution regulation system pH of 0.8 ~ 1.1mol/l is added
To 6.4 ~ 6.6,30 ~ 45min is stirred at 10 ~ 15 DEG C, with 1.6 ~ 2.0Kg/cm at 10 ~ 15 DEG C2Pressure carry out filters pressing, take
Supernatant, as crude extract；By mass, the amount ratio of enzymolysis liquid, ethyl alcohol, sodium chloride and diatomite is 1:0.08~0.12:
0.04~0.06:0.05~0.08；
Ethyl alcohol, sodium chloride, diatomite and wheat bran are added into crude extract, the aqueous hydrochloric acid solution that 0.8 ~ 1.1mol/l is added adjusts body
It is pH to 6.8 ~ 7.0,30 ~ 45min is stirred at 3 ~ 5 DEG C, with 1.6 ~ 2.0Kg/cm at 3 ~ 5 DEG C2Pressure carry out filters pressing, take
Precipitating；By mass, the amount ratio of crude extract, ethyl alcohol, sodium chloride, diatomite and wheat bran is 1:0.08~0.12:0.02~0.04:
0.03~0.05:0.04~0.06；
It takes and is deposited in 8 ~ 12hr of freezing at -30 ~ -20 DEG C, pulverized at 0 ~ 10 DEG C, water is added, stirs 30 at 10 ~ 15 DEG C
~ 45min filters and takes filtrate with 0.45 μm or 0.22 μm of organic filter film, at -30 ~ -20 DEG C freeze 8 ~ 12hr to get；It presses
The amount ratio of quality meter, precipitating and water is 1:2~3.
2. a kind of preparation method of low molecular polypeptide according to claim 1, which is characterized in that include the following steps：
（1）Pre-treatment
Fresh Goat Placenta is taken, removes fascia and blood vessel, washing, dry in the air 1.5hr at room temperature；It shears blocking, is freezed at -25 DEG C
10hr is pulverized at 4 DEG C and is obtained slurries；
（2）Enzymatic hydrolysis
Under stirring condition, water is added into slurries, the aqueous sodium carbonate regulation system pH to 7.8 of 1.0mol/l is added, is added
Trypsase and Protamex are warming up to 42 DEG C and are reacted, until enzymatic hydrolysis is completely, filtrate are taken after being filtered with filter paper, is as digested
Liquid；
By mass, the amount ratio of slurries, water, trypsase and Protamex are 1:5:0.3:0.15；
（3）It extracts
Ethyl alcohol, sodium chloride and diatomite are added into enzymolysis liquid, the aqueous hydrochloric acid solution regulation system pH of 1.0mol/l is added extremely
6.5,40min is stirred at 12 DEG C, with 1.8Kg/cm at 12 DEG C2Pressure carry out filters pressing, take supernatant, as crude extract；
By mass, the amount ratio of enzymolysis liquid, ethyl alcohol, sodium chloride and diatomite is 1:0.10:0.05:0.06；
Ethyl alcohol, sodium chloride, diatomite and wheat bran are added into crude extract, the aqueous hydrochloric acid solution regulation system pH of 1.0mol/l is added
To 6.9,40min is stirred at 4 DEG C, with 1.8Kg/cm at 4 DEG C2Pressure carry out filters pressing, take precipitating；By mass, it slightly mentions
Liquid, ethyl alcohol, sodium chloride, diatomite and wheat bran amount ratio be 1:0.10:0.03:0.04:0.05；
It takes to be deposited at -25 DEG C and freezes 10hr, pulverized at 4 DEG C, water is added, 40min is stirred at 12 DEG C, with 0.22 μm
Organic filter film filters and takes filtrate, at -25 DEG C freeze 10hr to get；By mass, it precipitates and the amount ratio of water is 1:
2.4。
3. a kind of preparation method of low molecular polypeptide according to claim 1 or 2, which is characterized in that step（3）Every step
The second alcohol and water of middle addition passes through the pre-heat treatment, and the temperature of preheating is consistent with the temperature that it is stirred to react.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971810A (en) * | 2019-04-10 | 2019-07-05 | 长沙嘉兰生物科技有限公司 | A kind of sheep embryo extract protein small peptide and its preparation process |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1242196A (en) * | 1998-07-17 | 2000-01-26 | 西安阳光保健品开发有限责任公司 | Method for preparing goat fetal hormone injection |
| WO2011085602A1 (en) * | 2010-01-14 | 2011-07-21 | 华南理工大学 | Protein hydrolysate, polypeptide solution and polypeptide, preparation method and use thereof |
| CN102329827A (en) * | 2011-07-12 | 2012-01-25 | 湖州康海斯生物科技有限公司 | Sheep placenta amino acid and low molecular weight polypeptide, and preparation method for sheep placenta amino acid and low molecular weight polypeptide |
| CN104372057A (en) * | 2014-11-17 | 2015-02-25 | 得利斯集团有限公司 | Extracting method of placenta |
| CN106727709A (en) * | 2016-11-24 | 2017-05-31 | 湖州珍露生物制品有限公司 | A kind of production technology of sheep placenta extract solution |
-
2018
- 2018-08-01 CN CN201810867788.4A patent/CN108866136A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1242196A (en) * | 1998-07-17 | 2000-01-26 | 西安阳光保健品开发有限责任公司 | Method for preparing goat fetal hormone injection |
| WO2011085602A1 (en) * | 2010-01-14 | 2011-07-21 | 华南理工大学 | Protein hydrolysate, polypeptide solution and polypeptide, preparation method and use thereof |
| CN102329827A (en) * | 2011-07-12 | 2012-01-25 | 湖州康海斯生物科技有限公司 | Sheep placenta amino acid and low molecular weight polypeptide, and preparation method for sheep placenta amino acid and low molecular weight polypeptide |
| CN104372057A (en) * | 2014-11-17 | 2015-02-25 | 得利斯集团有限公司 | Extracting method of placenta |
| CN106727709A (en) * | 2016-11-24 | 2017-05-31 | 湖州珍露生物制品有限公司 | A kind of production technology of sheep placenta extract solution |
Non-Patent Citations (3)
| Title |
|---|
| DIKE TENG: "Optimization of enzymatic hydrolysis parameters for antioxidant capacity of peptide from goat placenta", 《FOOD AND BIOPRODUCTS PROCESSING》 * |
| 朱蓓薇: "双酶水解法制备羊胎盘活性肽", 《食品与发酵工业》 * |
| 郑里翔: "《生物化学》", 31 January 2015 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971810A (en) * | 2019-04-10 | 2019-07-05 | 长沙嘉兰生物科技有限公司 | A kind of sheep embryo extract protein small peptide and its preparation process |
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