CN110205352A - A kind of preparation method of tree peony protein peptides - Google Patents
A kind of preparation method of tree peony protein peptides Download PDFInfo
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Abstract
The present invention relates to tree peony protein peptides preparation methods, and specific step is as follows, extract tree peony albumen from the peony seeds dregs of rice with kind skin or without kind of skin, utilize supercritical CO2Abstraction technique removes acid, selects suitable complex enzyme, passes through suitable combination, controlled enzymatic hydrolysis reaction condition destroys the peptide bond of different loci, can directly obtain the protein peptides of different specified molecular weights, specified molecular weight includes being less than 1000Da, 1000Da-2000Da, 2000Da-3000Da, the albumen peptide yield of specified molecular weight is all 95% or more, the method that the present invention prepares tree peony protein peptides is classified without ultrafiltration, reaction step is saved, preparation efficiency is improved, reduces production cost.
Description
Technical field
The present invention relates to peony seeds cake protein peptide technology field, in particular to a kind of preparation method of tree peony protein peptides.
Background technique
Tree peony is Ranunculaceae Paeonia shrub, be distributed widely in Luoyang, henan, Heze City, Shandong Province, Tongling, Anhui Province, Hanzhong,
The ground such as Hebei Bai Shan, with Luoyang, Heze, Tongling plantation at most, according to statistics, the cultivated area of China tree peony up to 300,000 mu, by
It is high in the oil content of peony seeds, and it is rich in linoleic acid plus linolenic acid, therefore in recent years, peony seeds are widely used in oil expression, and
The solid residue peony seeds dregs of rice after oil expression, are directly discarded mostly or as fertilizer, utilization power is undesirable, are sent out by measurement
Existing, crude protein content is about 20% in the peony seeds dregs of rice, therefore, extracts peony seeds using the peony seeds dregs of rice for extracting peony seed oil
Albumen is possibly realized.
Protein is the important composition ingredient of food, and protein obtains protein peptides, functional characteristic and biology after digesting
Characteristic is learned, has bigger difference with original protein, the dissolubility as protein can be improved after digesting, emulsibility, the function such as foaming characteristic
Can characteristic, there is a peony seeds dregs of rice product after a large amount of oil refining in China every year, available a large amount of tree peony albumen, therefore, how
High efficiency, low cost obtain tree peony protein peptides needed for meeting life, are present research hotspots.
Summary of the invention
It is an object of the invention to overcome the above problem, the method that specified molecular weight tree peony protein peptides can be obtained is developed,
This method preparation safety, simple process are not had to be classified using ultrafiltration, improve preparation efficiency, reduce production cost.
The purpose of the present invention is achieved by the following technical programs:
A kind of preparation method of tree peony protein peptides, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:20~30 with water,
NaOH is added and adjusts pH9~10,60~70min of magnetic agitation under the conditions of 50~60 DEG C, be centrifuged (3500~4000r/min, 13
~18min), supernatant is taken, HCl is added, adjusts pH3~4, is centrifuged (3500~4000r/min, 13~18min), precipitating is used few
Amount deionized water is freeze-dried after cleaning to neutrality, obtains peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 10-14MPa, extraction
100~120min, room temperature stands half an hour after having extracted, and collects protein liquid, can deviate from acid in tree peony albumen；
S3: taking obtained by step S2 3 parts of protein liquid, and compound protease is added and is digested, the compound protease includes pancreas
1~2 part of chrymotrypsin, 3~5 parts of pectase, enzyme activity is 110~120u/mL, enzyme digestion reaction pH6.5~7.0, reaction temperature
40~60 DEG C, 2~4h of reaction time of degree, after enzymatic hydrolysis, 8%~12% HCl is added in high-temperature inactivation, adjusts pH to 4.0~4.5,
It is stirred under the conditions of 90~100r/min, the tree peony albumen not digested is precipitated in curdy, is separated by filtration, it is spraying after purification
Dry that tree peony protein peptides, SDS-PAGE electrophoretic determination protein relative molecular mass are less than 1000Da, Kjeldahl's method is surveyed
Surely the yield less than 1000Da protein peptides is obtained 95% or more.
Preferably, the compound protease in the step S3 is beta galactosidase and a- glucuroide, and β-half is added
1~2 part of lactoside enzyme, 4~5 parts of a- glucuroide, enzyme activity is 80~90u/mL, and enzyme digestion reaction pH is 6.0~7.0, instead
Answer 40~45 DEG C of temperature, 3~4h of reaction time, after enzymatic hydrolysis, 8%~12% HCl is added in high-temperature inactivation, adjusts pH to 4.0
~4.5, it is stirred under the conditions of 70~80r/min, the tree peony albumen not digested is precipitated in curdy, is separated by filtration, after purification
Tree peony protein peptides are spray-dried to obtain, SDS-PAGE electrophoretic determination protein relative molecular mass is 1000~2000Da, and kelvin is fixed
Nitrogen method measures to obtain the yield of 1000~2000Da protein peptides 95% or more.
Preferably, the step S3 compound protease is 1~2 part of chymotrypsin, and 3~5 parts of pectase, enzyme activity is
110~120u/mL, enzyme digestion reaction pH6.5~7.0,40~60 DEG C of reaction temperature, 2~4h of reaction time, after enzymatic hydrolysis, high temperature goes out
It is living, 8%~12% HCl is added, adjusts pH to 4.0~4.5, is stirred under the conditions of 90~100r/min, make not digest male
Red albumen is precipitated in curdy, is separated by filtration, tree peony protein peptides, SDS-PAGE electrophoretic determination protein are spray-dried to obtain after purification
Relative molecular mass is 2000~3000Da, Kjeldahl nitrogen determination obtain the yield of 2000~3000Da protein peptides 95% with
On.
Different complex enzyme combinations, can destroy the peptide bond of tree peony albumen different loci, different specific so as to obtain
The tree peony protein peptides of molecular weight.
Preferably, the peony seeds dregs of rice are the peony seeds dregs of rice with kind skin, or the peony seeds dregs of rice without kind of skin.
Preferably, 80~100 meshes are crossed in the S1.
Preferably, the S3 enzyme digestion reaction pH is 6.8, and reaction temperature is 58 DEG C, reaction time 2.5h, 95 DEG C after enzymatic hydrolysis
High-temperature inactivation 15min.
Preferably, when the compound protease is beta galactosidase and a- glucuroide, the enzyme digestion reaction pH is
6.5, reaction temperature is 43 DEG C, reaction time 3.5h, 95 DEG C of high-temperature inactivation 15min after enzymatic hydrolysis.
Preferably, when the compound protease is cellulase and carbohydrase, the enzyme digestion reaction pH is 6.0, reaction temperature
Degree is 50 DEG C, reaction time 3.5h, 95 DEG C of high-temperature inactivation 15min after enzymatic hydrolysis.
The present invention, which has following technical effect that, compared with prior art recycles the peony seeds dregs of rice, the peony seeds
The dregs of rice are band kind skin, or without kind of skin, first prepare tree peony protein isolate, recycling supercritical CO2Technology extraction, removes male
Acid in red albumen, gained tree peony albumen chymotrypsin and pectin enzyme reaction, available specified molecular weight are less than
The tree peony protein peptides of 1000Da, yield is 95% or more；Gained tree peony albumen beta galactosidase and a- glucuroide are anti-
It answers, available specified molecular weight is the tree peony protein peptides of 1000Da-2000Da, and yield is 95% or more；Gained tree peony albumen
With cellulase and saccharification enzyme reaction, available specified molecular weight is the tree peony protein peptides of 2000Da-3000Da, and yield exists
95% or more.
Specific embodiment
Present invention will be further explained below with reference to specific examples.These embodiments are merely to illustrate the present invention and do not have to
In limiting the scope of the invention.Test method without specific conditions in lower example embodiment, usually according to this field conventional strip
Part or the condition suggested according to manufacturer.Any unsubstantiality that those skilled in the art is done on the basis of the present invention
Variation and replacement belong to scope of the present invention.
Embodiment 1
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 12MPa, extraction
110min, room temperature stands half an hour after having extracted, and collects protein liquid；
S3: taking obtained by step S2 3 parts of protein liquid, and compound protease is added and is digested, the compound protease includes pancreas
1 part of chrymotrypsin, 3 parts of pectase, enzyme activity 115u/mL, enzyme digestion reaction pH6.5,55 DEG C of reaction temperature, the reaction time
2.5h, after enzymatic hydrolysis, 10% HCl is added in high-temperature inactivation, is adjusted pH to 4.3, is stirred, be separated by filtration under the conditions of 90r/min,
Be spray-dried after purification relative molecular mass be less than 1000Da peony seeds protein peptides, yield 96.5%.
Embodiment 2
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 12MPa, extraction
110min, room temperature stands half an hour after having extracted, and collects protein liquid；
S3: taking obtained by step S2 3 parts of protein liquid, and compound protease is added and is digested, the compound protease includes pancreas
1.5 parts of chrymotrypsin, 3.5 parts of pectase, enzyme activity 118u/mL, enzyme digestion reaction pH6.8,58 DEG C of reaction temperature, when reaction
Between 2.5h, after enzymatic hydrolysis, 10% HCl is added in high-temperature inactivation, adjust pH to 4.3, stirred under the conditions of 90r/min, filtering point
From, be spray-dried after purification relative molecular mass be less than 1000Da peony seeds protein peptides, yield 96.9%.
Embodiment 3
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 12MPa, extraction
110min, room temperature stands half an hour after having extracted, and collects protein liquid；
S3: taking obtained by step S2 3 parts of protein liquid, and compound protease is added and is digested, the compound protease includes pancreas
2 parts of chrymotrypsin, 4 parts of pectase, enzyme activity 114u/mL, enzyme digestion reaction pH7.0,60 DEG C of reaction temperature, the reaction time
3.0h, after enzymatic hydrolysis, 10% HCl is added in high-temperature inactivation, is adjusted pH to 4.3, is stirred, be separated by filtration under the conditions of 90r/min,
Be spray-dried after purification relative molecular mass be less than 1000Da peony seeds protein peptides, yield 95.8%.
Embodiment 4
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 12MPa, extraction
110min, room temperature stands half an hour after having extracted, and collects protein liquid；
Compound protease in step S3 is beta galactosidase and a- glucuroide, and β-galactosidase 1 part is added,
4 parts of a- glucuroide, enzyme activity 87u/mL, enzyme digestion reaction pH are 6.5,45 DEG C of reaction temperature, reaction time 3h, are digested
Afterwards, 10% HCl is added in high-temperature inactivation, adjusts pH to 4.6, stirs, be separated by filtration under the conditions of 75r/min, spraying after purification
Dry 1000~2000Da peony seeds protein peptides, yield 96.3%.
Embodiment 5
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 12MPa, extraction
110min, room temperature stands half an hour after having extracted, and collects protein liquid；
Compound protease in step S3 is beta galactosidase and a- glucuroide, and β-galactosidase 1 .5 is added
Part, 4.5 parts of a- glucuroide, enzyme activity 89u/mL, enzyme digestion reaction pH be 6.5,45 DEG C of reaction temperature, the reaction time
3.5h, after enzymatic hydrolysis, 10% HCl is added in high-temperature inactivation, is adjusted pH to 4.6, is stirred, be separated by filtration under the conditions of 75r/min,
1000~2000Da peony seeds protein peptides, yield 95.8% are spray-dried to obtain after purification.
Embodiment 6
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 12MPa, extraction
110min, room temperature stands half an hour after having extracted, and collects protein liquid；
Compound protease in step S3 is beta galactosidase and a- glucuroide, and β-galactosidase 2 part is added,
5 parts of a- glucuroide, enzyme activity 86u/mL, enzyme digestion reaction pH are 7.0,45 DEG C of reaction temperature, reaction time 4h, are digested
Afterwards, 10% HCl is added in high-temperature inactivation, adjusts pH to 4.6, stirs, be separated by filtration under the conditions of 75r/min, spraying after purification
Dry 1000~2000Da peony seeds protein peptides, yield 96.2%.
Embodiment 7
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 13MPa, extraction
105min, room temperature stands half an hour after having extracted, and collects protein liquid；
Compound protease in step S3 is cellulase and carbohydrase, is added 1 part of cellulase, 5 parts of carbohydrase, enzyme activity
Power is 99u/mL, enzyme digestion reaction pH5.0, and 50 DEG C of reaction temperature, reaction time 3.0h, after enzymatic hydrolysis, high-temperature inactivation is added 10%
HCl adjusts pH to 4.6, stirs, be separated by filtration under the conditions of 60r/min, 2000~3000Da egg is spray-dried to obtain after purification
White peptide, yield 96.4%.
Embodiment 8
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 13MPa, extraction
105min, room temperature stands half an hour after having extracted, and collects protein liquid；
Compound protease in step S3 is cellulase and carbohydrase, is added 2 parts of cellulase, 5.5 parts of carbohydrase, enzyme
Vigor is 103u/mL, enzyme digestion reaction pH6.0, and 50 DEG C of reaction temperature, reaction time 4.0h, after enzymatic hydrolysis, high-temperature inactivation is added
10% HCl, adjust pH to 4.6, stir, be separated by filtration under the conditions of 60r/min, be spray-dried after purification 2000~
3000Da protein peptides, yield 95.3%.
Embodiment 9
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 13MPa, extraction
105min, room temperature stands half an hour after having extracted, and collects protein liquid；
Compound protease in step S3 is cellulase and carbohydrase, is added 3 parts of cellulase, 6 parts of carbohydrase, enzyme activity
Power is 98u/mL, enzyme digestion reaction pH7.0, and 50 DEG C of reaction temperature, reaction time 4.0h, after enzymatic hydrolysis, high-temperature inactivation is added 10%
HCl adjusts pH to 4.6, stirs, be separated by filtration under the conditions of 60r/min, 2000~3000Da egg is spray-dried to obtain after purification
White peptide, yield 96.2%.
Comparative example 1
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 12MPa, extraction
110min, room temperature stands half an hour after having extracted, and collects protein liquid；
S3: taking obtained by step S2 3 parts of protein liquid, and compound protease is added and is digested, the compound protease includes pancreas
0.5 part of chrymotrypsin, 2 parts of pectase, enzyme activity 43u/mL, enzyme digestion reaction pH6.8,55 DEG C of reaction temperature, the reaction time
2.5h, after enzymatic hydrolysis, 10% HCl is added in high-temperature inactivation, is adjusted pH to 4.3, is stirred, be separated by filtration under the conditions of 90r/min,
Be spray-dried after purification relative molecular mass be less than 1000Da peony seeds protein peptides, yield 47.1%.
Comparative example 2
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 12MPa, extraction
110min, room temperature stands half an hour after having extracted, and collects protein liquid；
S3: taking obtained by step S2 3 parts of protein liquid, and compound protease is added and is digested, the compound protease includes pancreas
1 part of chrymotrypsin, 2 parts of pectase, enzyme activity 39u/mL, enzyme digestion reaction pH6.0,55 DEG C of reaction temperature, the reaction time
2.0h after enzymatic hydrolysis, 10% HCl is added in high-temperature inactivation, pH to 4.3 is adjusted, stirs, is separated by filtration under the conditions of 90r/min,
Be spray-dried after purification relative molecular mass be less than 1000Da peony seeds protein peptides, yield 49.3%.
Comparative example 3
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 12MPa, extraction
110min, room temperature stands half an hour after having extracted, and collects protein liquid；
S3: taking obtained by step S2 3 parts of protein liquid, and compound protease is added and is digested, the compound protease includes pancreas
3 parts of chrymotrypsin, 7 parts of pectase, enzyme activity 49u/mL, enzyme digestion reaction pH6.5,55 DEG C of reaction temperature, the reaction time
3.0h, after enzymatic hydrolysis, 10% HCl is added in high-temperature inactivation, is adjusted pH to 4.3, is stirred, be separated by filtration under the conditions of 90r/min,
Be spray-dried after purification relative molecular mass be less than 1000Da peony seeds protein peptides, yield 52.7%.
Comparative example 4
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 12MPa, extraction
110min, room temperature stands half an hour after having extracted, and collects protein liquid；
Compound protease in step S3 is beta galactosidase and a- glucuroide, and β-galactosidase 1 part is added,
2 parts of a- glucuroide, enzyme activity 29u/mL, enzyme digestion reaction pH are 6.0,45 DEG C of reaction temperature, reaction time 3h, are digested
Afterwards, 10% HCl is added in high-temperature inactivation, adjusts pH to 4.6, stirs, be separated by filtration under the conditions of 75r/min, spraying after purification
Dry 1000~2000Da peony seeds protein peptides, yield 47.5%.
Comparative example 5
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 12MPa, extraction
110min, room temperature stands half an hour after having extracted, and collects protein liquid；
Compound protease in step S3 is beta galactosidase and a- glucuroide, and β-galactosidase 2 part is added,
1 part of a- glucuroide, enzyme activity 33u/mL, enzyme digestion reaction pH are 6.0,45 DEG C of reaction temperature, reaction time 4.0h, are digested
Afterwards, 10% HCl is added in high-temperature inactivation, adjusts pH to 4.6, stirs, be separated by filtration under the conditions of 75r/min, spraying after purification
Dry 1000~2000Da peony seeds protein peptides, yield 51.7%.
Comparative example 6
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 12MPa, extraction
110min, room temperature stands half an hour after having extracted, and collects protein liquid；
Compound protease in step S3 is beta galactosidase and a- glucuroide, and β-galactosidase 2 part is added,
7 parts of a- glucuroide, enzyme activity 33u/mL, enzyme digestion reaction pH are 7.5,45 DEG C of reaction temperature, reaction time 4.0h, are digested
Afterwards, 10% HCl is added in high-temperature inactivation, adjusts pH to 4.6, stirs, be separated by filtration under the conditions of 75r/min, spraying after purification
Dry 1000~2000Da peony seeds protein peptides, yield 49.3%.
Comparative example 7
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 13MPa, extraction
105min, room temperature stands half an hour after having extracted, and collects protein liquid；
Compound protease in step S3 is cellulase and carbohydrase, is added 1 part of cellulase, 3 parts of carbohydrase, enzyme activity
Power is 41u/mL, enzyme digestion reaction pH7.0, and 50 DEG C of reaction temperature, reaction time 3.0h, after enzymatic hydrolysis, high-temperature inactivation is added 10%
HCl adjusts pH to 4.6, stirs, be separated by filtration under the conditions of 60r/min, 2000~3000Da egg is spray-dried to obtain after purification
White peptide, yield 29.6%.
Comparative example 8
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 13MPa, extraction
105min, room temperature stands half an hour after having extracted, and collects protein liquid；
Compound protease in step S3 is cellulase and carbohydrase, is added 2 parts of cellulase, 4 parts of carbohydrase, enzyme activity
Power is 43u/mL, enzyme digestion reaction pH7.0, and 50 DEG C of reaction temperature, reaction time 4.0h, after enzymatic hydrolysis, high-temperature inactivation is added 10%
HCl adjusts pH to 4.6, stirs, be separated by filtration under the conditions of 60r/min, 2000~3000Da egg is spray-dried to obtain after purification
White peptide, yield 36.5%.
Comparative example 9
A kind of tree peony protein peptides preparation method, the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:25 with water, is added
NaOH adjusts pH9.35, and magnetic agitation 66min under the conditions of 57.6 DEG C is centrifuged (3800r/min, 15min), takes supernatant, is added
HCl adjusts pH3.5, is centrifuged (3800r/min, 15min), and precipitating is freeze-dried after being cleaned with a small amount of deionized water to neutrality, obtains
Peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 13MPa, extraction
105min, room temperature stands half an hour after having extracted, and collects protein liquid；
Compound protease in step S3 is cellulase and carbohydrase, is added 4 parts of cellulase, 7 parts of carbohydrase, enzyme activity
Power is 38u/mL, enzyme digestion reaction pH8.0, and 50 DEG C of reaction temperature, reaction time 4.0h, after enzymatic hydrolysis, high-temperature inactivation is added 10%
HCl adjusts pH to 4.6, stirs, be separated by filtration under the conditions of 60r/min, 2000~3000Da egg is spray-dried to obtain after purification
White peptide, yield 39.8%.
Experimental example 1
Kjeldahl nitrogen determination albumen peptide yield
(1) nitrogen content in protein is measured
By protein and concentrated sulfuric acid heat together, catalyst sulfuric acid copper is added thereto, sulfuric acid decomposition is sulfur dioxide, water and original
Sub- state oxygen, protein oxidation is carbon dioxide and water, and nitrogen therein changes ammonification, and further generates ammonium sulfate；It has reacted
NaOH is added after complete, NH4+ is changed into NH3, and NH3 is imported in excessive boric acid solution by distilling, is dripped by standard hydrochloric acid
Fixed, the standard hydrochloric acid molal quantity for titrating consumption is the molal quantity of NH3, i.e., the content M of nitrogen in protein.
(2) nitrogen content in protein peptides is measured
By protein peptides and concentrated sulfuric acid heat together, catalyst sulfuric acid copper is added thereto, sulfuric acid decomposition is sulfur dioxide, water and original
Sub- state oxygen, protein oxidation is carbon dioxide and water, and nitrogen therein changes ammonification, and further generates ammonium sulfate；It has reacted
NaOH is added after complete, NH4+ is changed into NH3, and NH3 is imported in excessive boric acid solution by distilling, is dripped by standard hydrochloric acid
Fixed, the standard hydrochloric acid molal quantity for titrating consumption is the molal quantity of NH3, the content m of nitrogen as in protein peptides.
Nitrogen content M*100% in nitrogen content m/ protein in albumen peptide yield=protein peptides
Experimental example 2
SDS-PAGE electrophoretic determination protein relative molecular mass
(1) instrument, raw materials and reagents
Instrument: vertical version type electrophoresis tank；D.C. regulated power supply；50uL pipette tips, glass plate, water-bath, staining trough, beaker,
Pipette, dropper.
Raw material: 0.5mg/mL sample solution
Reagent: Tris-HCl pH of buffer 8.9, Tris-HCl pH of buffer 6.7,30% separation gel liquid storage, 10% concentration
Glue liquid storage, 10%SDS solution, dyeing liquor, destainer.
(2) experimental procedure
1, electrophoresis tank is installed
2, match glue
3, gel slab is prepared
4, sample treatment and sample-adding
0.5mg/mL sample solution and standard protein liquid boiling water bath are heated 3 minutes, be cooled to room temperature it is spare, by 15uL
Sample is carefully added to gel spill sample trench bottom, to all add sample in all spill sample cells, starts electrophoresis.
5, electrophoresis
Electrophoresis apparatus is opened, electric current is transferred to 10mA.When sample separation, electric current is adjusted to 25mA, when blue dyes migrates
To bottom, electric current is set to zero, and closes power supply, pulls out fixed plate, takes out glass plate, one block of glass is gently pried open shifting with blade
It goes, makes marks in offset plate one end, dye.
6, it dyes and decolourizes
Dyeing liquor is poured into culture dish, dyeing 1h or so for several times, then with destainer is decolourized with distilled water rinsing, until
Zone of protein is clear, measures each band at a distance from gel top with ruler.
7, it calculates
It is mapped with the logarithm of standard protein molecular weight to relative mobility, standard curve is obtained, according to sample to be tested phase
To mobility, its molecular weight can be found out from standard curve.
Relative mobility mR=sample migration distance (cm)/dye migration distance (cm)
Under embodiment 1-3 and comparative example 1-3 reaction condition, chymotrypsin and pectase reaction result are as shown in table 1:
Under 1 chymotrypsin of table and pectase reaction condition, 3 parts of tree peony albumen are added, are less than 1000Da tree peony albumen
The preparation of peptide
Under embodiment 4-6 and comparative example 4-6 reaction condition, beta galactosidase and a- glucuroide reaction result such as table
Shown in 2:
Under 2 beta galactosidase of table and a- glucuroide reaction condition, 3 parts of tree peony albumen, 1000~2000Da is added
The preparation of tree peony protein peptides
Under embodiment 7-9 and comparative example 7-9 reaction condition, cellulase and carbohydrase reaction result are as shown in table 3:
Under 3 cellulase of table and carbohydrase reaction condition, 3 parts of tree peony albumen, 2000`3000Da tree peony protein peptides are added
Preparation
The word of such as "include", "comprise", " having " involved in the disclosure etc. is open vocabulary, refer to " including
But it is not limited to ", and can be used interchangeably with it.Vocabulary "or" and "and" used herein above refer to vocabulary "and/or", and can be with it mutually
Use is changed, unless it is not such that context, which is explicitly indicated,.Vocabulary " such as " used herein above refers to phrase " such as, but not limited to ",
And it can be used interchangeably with it.
In addition, as used herein, the "or" instruction separation used in the enumerating of the item started with "at least one"
Enumerate, for example, " at least one of A, B or C " enumerate mean A or B or C or AB or AC or BC or ABC (i.e. A and B and
C).In addition, wording " exemplary " does not mean that the example of description is preferred or more preferable than other examples.
The above description of disclosed aspect is provided so that any person skilled in the art can make or use this
It is open.Various modifications in terms of these are readily apparent to those skilled in the art, and are defined herein
General Principle can be applied to other aspect without departing from the scope of the present disclosure.Therefore, the disclosure is not intended to be limited to
Aspect shown in this, but according to principle disclosed herein and the consistent widest range of novel feature.
Above description is had been presented for for purposes of illustration and description.In addition, this description is not intended to the reality of the disclosure
It applies example and is restricted to form disclosed herein.Although already discussed above multiple exemplary aspects and embodiment, this field skill
Its certain modifications, modification, change, addition and sub-portfolio will be recognized in art personnel.
Claims (8)
1. a kind of preparation method of tree peony protein peptides, which is characterized in that the method step are as follows:
S1: the peony seeds dregs of rice are crushed, supercritical CO is used2Degreasing is extracted, sieving is mixed by solid-liquid ratio 1:20~30 with water, is added
NaOH adjusts pH9~10,60~70min of magnetic agitation under the conditions of 50~60 DEG C, be centrifuged (3500~4000r/min, 13~
18min), supernatant is taken, HCl is added, adjusts pH3~4, is centrifuged (3500~4000r/min, 13~18min), precipitating is used a small amount of
Deionized water is freeze-dried after cleaning to neutrality, obtains peony seeds albumen；
S2: by peony seeds protein dissolution obtained by S1, lysate supercritical CO2Extraction, extracting pressure 10-14MPa, extraction 100~
120min, room temperature stands half an hour after having extracted, and collects protein liquid；
S3: taking obtained by step S2 3 parts of protein liquid, and compound protease is added and is digested, the compound protease includes pancreas curdled milk
1~2 part of protease, 3~5 parts of pectase, enzyme activity is 110~120u/mL, enzyme digestion reaction pH6.5~7.0, reaction temperature 40
~60 DEG C, 2~4h of reaction time, after enzymatic hydrolysis, 8%~12% HCl is added in high-temperature inactivation, pH to 4.0~4.5 is adjusted, 90
It stirs, is separated by filtration under the conditions of~100r/min, tree peony protein peptides are spray-dried to obtain after purification.
2. the preparation method of tree peony protein peptides according to claim 1, which is characterized in that the compound protein in the step S3
Enzyme be beta galactosidase and a- glucuroide, be added β-galactosidase 1~2 part, 4~5 parts of a- glucuroide, enzyme activity
Power is 80~90u/mL, and enzyme digestion reaction pH is 6.0~7.0,40~45 DEG C of reaction temperature, 3~4h of reaction time, high after enzymatic hydrolysis
Temperature inactivation, is added 8%~12% HCl, adjusts pH to 4.0~4.5, stirs, be separated by filtration under the conditions of 70~80r/min,
Tree peony protein peptides are spray-dried to obtain after purification.
3. the preparation method of tree peony protein peptides according to claim 1, which is characterized in that the compound protein in the step S3
Enzyme is cellulase and carbohydrase, is added 1~3 part of cellulase, 5~6 parts of carbohydrase, enzyme activity is 95~105u/mL, enzymatic hydrolysis
PH5.0~7.0 are reacted, 48~53 DEG C of reaction temperature, 3~4h of reaction time, after enzymatic hydrolysis, high-temperature inactivation is added 8%~12%
HCl adjusts pH to 4.0~4.5, stirs, be separated by filtration under the conditions of 55~65r/min, tree peony egg is spray-dried to obtain after purification
White peptide.
4. according to claim 1, the preparation method of the 2 or 3 tree peony protein peptides, which is characterized in that the peony seeds dregs of rice are band
The peony seeds dregs of rice of kind skin, or the peony seeds dregs of rice without kind of skin.
5. the preparation method of tree peony protein peptides according to claim 1, which is characterized in that cross 80~100 meshes in the S1.
6. the preparation method of the tree peony protein peptides according to claim 1, it is characterised in that: the S3 enzyme digestion reaction
PH6.8, reaction temperature are 58 DEG C, reaction time 2.5h, 95 DEG C of high-temperature inactivation 15min after enzymatic hydrolysis.
7. the preparation method of the tree peony protein peptides according to claim 2, it is characterised in that: the enzyme digestion reaction pH is
6.5, reaction temperature is 43 DEG C, reaction time 3.5h, 95 DEG C of high-temperature inactivation 15min after enzymatic hydrolysis.
8. the preparation method of the tree peony protein peptides according to claim 3, it is characterised in that: the enzyme digestion reaction pH is
6.0, reaction temperature is 50 DEG C, reaction time 3.5h, 95 DEG C of high-temperature inactivation 15min after enzymatic hydrolysis.
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| CN111072753A (en) * | 2020-01-14 | 2020-04-28 | 广州泽力医药科技有限公司 | Low-temperature extraction method of high-purity protein in peony seed meal |
| CN112891509A (en) * | 2021-01-28 | 2021-06-04 | 衍生健康医药(广东)有限公司 | Seven-star tea granules and preparation method thereof |
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| CN108998491A (en) * | 2018-08-29 | 2018-12-14 | 西南大学 | A method of extracting protein in the peony seeds dregs of rice |
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