CN108342443A - A kind of method that anti-oxidation peptide is extracted in the peony seeds dregs of rice - Google Patents
A kind of method that anti-oxidation peptide is extracted in the peony seeds dregs of rice Download PDFInfo
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- CN108342443A CN108342443A CN201810377122.0A CN201810377122A CN108342443A CN 108342443 A CN108342443 A CN 108342443A CN 201810377122 A CN201810377122 A CN 201810377122A CN 108342443 A CN108342443 A CN 108342443A
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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
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- 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
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
Abstract
The present invention relates to a kind of methods that anti-oxidation peptide is extracted in peony seeds, belong to technical field of agricultural product process.This method is realized especially by following steps：Microwave thawing after the peony seeds dregs of rice are freezed, drying and grinding is at powder after degreasing；Solvent, lactitol and citric acid is added in peony seeds dregs of rice powder, adjusts pH value to 11.0,1 ultrasounds after standing, centrifuging and taking supernatant, then adjusting pH to 3.80, centrifuges, and precipitation freeze-drying obtains freeze-dried powder；It weighs freeze-dried powder and is dissolved in deionized water, beeswax is added, preheating obtains mixed liquor；It is 8.0 that mixed liquor, which is adjusted pH, and protease, enzymolysis is added, and enzyme deactivation is lived, and collects supernatant freeze-drying.The present invention is raw material using the peony seeds dregs of rice, consider degree of hydrolysis and inoxidizability, obtain and prepare the optimised process having compared with strong anti-oxidative activity polypeptide using peony seeds cake protein, theoretical foundation is provided for the intensive processing utilization of the peony seeds dregs of rice, and preparation process is simple, market prospects are good.
Description
Technical field
The present invention relates to a kind of methods that anti-oxidation peptide is extracted in peony seeds, belong to technical field of agricultural product process.
Background technology
Biologically active peptide is the peptides between protein and amino acid, with desired bioactivity, including it is anti-
Oxidation, blood pressure lowering, antithrombus formation, reducing blood lipid, antimicrobial and anti-inflammatory effect.Anti-oxidation peptide is by 5 ~ 16 amino acid residue groups
At compared with other biomolecule, with extremely strong bioactivity and diversity, inoxidizability is more notable, safe.Mesh
Before to be used to prepare the method for anti-oxidation peptide mainly include artificial synthesized method, enzymatic isolation method and biological fermentation process, preparing bioactivity
During peptide, zymolysis technique is most fast, most safe, most manageable technology, it can improve simultaneously protein functionality and
Biological characteristics can also increase the commercial value of byproduct.
Tree peony belongs to Paeoniaceae Paeonia, is perennial machaka, be a kind of economic value and nutritive value all very
High plant.Peony seeds can be used for extracting oil, and seed oil is rich in alpha-linolenic acid, have the plurality of health care functions such as lowering blood pressure and blood fat；
Contain the abundant root bark of tree peony in moutan root bark, can be used for producing Chinese medicine；Flower, flower bud, the bud of tree peony can be used for making health protection tea；Peony seeds
Grouts are rich in dietary fiber, can be used for fermenting and producing ferment, adjust gastrointestinal health.It is from health ministry approval peony seed oil
Since new resource food, oil is drastically expanded with tree peony cultivated area, and peony seeds yield improves year by year, is generated after grease extraction male
The red seedcake dregs of rice are consequently increased.These industrial grouts are mainly as feedstuff or culture medium of edible fungus base-material, comprehensive exploitation
It is low using level；It is concentrated mainly on the optimal extraction technology and properties evaluations of peony seeds cake protein in terms of scientific research.Peony seeds
Cake protein content about 25%, but be also rarely reported currently with the research of the peony seeds cake protein of preparation research and development active peptide, individually
Process optimization in relation to peony seeds proteolysis is mostly to improve degree of hydrolysis as target.Due between degree of hydrolysis and oxidation resistance simultaneously
It is not simple linear relationship, the activity of the anti-oxidation peptide prepared according to protein hydrolysis degree is not also very high.
Invention content
In view of the problems of the existing technology, the present invention provides a kind of methods that anti-oxidation peptide is extracted in peony seeds dregs of rice.
Used technical solution is the present invention to achieve the goals above：
The present invention provides a kind of methods that anti-oxidation peptide is extracted in dregs of rice from peony seeds, include the following steps：
（1）Microwave thawing after the peony seeds dregs of rice are freezed, it is spare to obtain peony seeds dregs of rice powder at powder for drying and grinding after degreasing；
（2）By peony seeds dregs of rice powder according to volume ratio 1：Solvent is added in 25 mass ratioes, and lactitol and citric acid, mixing are added in slurry
After uniformly, 15-20min is stood, then addition NaOH solution tune pH value to 11.0,180 W, 2.5 h of ultrasound, centrifuging and taking supernatant,
Then pH to 3.80 is adjusted with HCl solution, centrifugation goes supernatant to stay precipitation, and precipitation, which is placed in vacuum freeze drier, to be lyophilized, and must be frozen
- 20 DEG C of dry powder stores for future use；
（3）It weighs freeze-dried powder and is dissolved in deionized water, beeswax is added, is placed in 80 DEG C of water-baths and preheats 20 min, obtain mixed liquor；
（4）It is 8.0 that mixed liquor, which is adjusted pH, and alkali protease and flavor protease is added, then enzymolysis liquid is placed in boiling by enzymolysis
It heats 10min enzyme deactivations in water to live, ice bath cools down rapidly, centrifuges, and collects supernatant freeze-drying.
Further, step（1）In, the peony seeds dregs of rice are freezed at -20 DEG C, then microwave thawing.
Further, the solvent is the mixed solution of deionized water and polyethylene glycol；The deionized water and poly- second two
The volume ratio of alcohol is 1：0.1-0.2.
Further, the addition of the lactitol accounts for the 1-2% of slurry gross weight；The addition of the citric acid accounts for slurry
The 0.8-1% of gross weight.
NaOH solution used in the present invention and the concentration of HCl solution are 1mol/L.
Further, step（3）In, 100mL deionized waters are added in the freeze-dried powder per 0.7g.
Further, the addition of the beeswax accounts for 0.05-0.08 times of freeze-dried powder weight.
Alkali protease and flavor protease used in the present invention are commercially available, the alkali protease and flavor albumen
The addition of enzyme is the 2.5% of mixed liquor weight；The mass ratio of the alkali protease and flavor protease is 1.5：1.
Further, the temperature of the enzymolysis is 56 DEG C, enzymolysis time 2h.
Degreasing used in the present invention is the degreasing method of routine.
Beneficial effects of the present invention are：
（1）The present invention is raw material using the peony seeds dregs of rice, considers degree of hydrolysis and inoxidizability, obtains and utilize peony seeds dregs of rice egg
It is white to prepare the optimised process having compared with strong anti-oxidative activity polypeptide, for the peony seeds dregs of rice intensive processing utilization provide theory according to
According to, and preparation process is simple, market prospects are good.
（2）Anti-oxidation peptide yield prepared by the present invention is high, purity is good.
（3）The peony seeds dregs of rice are subjected to redevelopment utilization, the comprehensive development and utilization for improving peony seeds grouts is horizontal.
Description of the drawings
Fig. 1 is the hydrolysis curves of different protease hydrolyzeds in peony seeds protein antioxidant peptide preparation process of the present invention
Figure.
Specific implementation mode
Below by specific embodiment, further explanation and description of the technical solution of the present invention are carried out.
Embodiment 1
（1）Microwave thawing after the peony seeds dregs of rice are freezed at -20 DEG C, it is spare to obtain peony seeds dregs of rice powder at powder for drying and grinding after degreasing；
（2）By peony seeds dregs of rice powder according to volume ratio 1：The mixed solution of deionized water and polyethylene glycol is added in 25 mass ratioes（Go from
The volume ratio of sub- water and polyethylene glycol is 1：0.1）Slurry is obtained after stirring evenly, and the lactitol and 1% for accounting for slurry gross weight 1.5% is added
Citric acid, after mixing, stand 15min, then be added 1mol/L NaOH solution tune pH value to 11.0,180 W ultrasound
Then 2.5 h, centrifuging and taking supernatant use the HCl solution of 1mol/L to adjust pH to 3.80, centrifugation goes supernatant that precipitation, precipitation is stayed to set
It is lyophilized in vacuum freeze drier, obtains -20 DEG C of freeze-dried powder and store for future use；
（3）It weighs freeze-dried powder 7g and is dissolved in 1000mL deionized waters, 0.35g beeswax powders are added, is placed in 80 DEG C of water-baths and preheats 20
Min obtains mixed liquor；
（4）It is 8.0 that mixed liquor is adjusted pH using the NaOH solution of 1mol/L, and the basic protein for accounting for mixed liquor weight 2.5% is added
Enzyme and flavor protease（The two ratio 1.5：1）, digest 2h in 56 DEG C of thermostat water bath, in enzymolysis process, utilize NaOH
Solution makes pH of mixed remain unchanged, and enzymolysis liquid is placed in boiling water after enzymolysis and heats 10min enzyme deactivations work, ice bath is cold rapidly
But, it centrifuges, collects supernatant freeze-drying.
Embodiment 2
（1）Microwave thawing after the peony seeds dregs of rice are freezed at -20 DEG C, it is spare to obtain peony seeds dregs of rice powder at powder for drying and grinding after degreasing；
（2）By peony seeds dregs of rice powder according to volume ratio 1：The mixed solution of deionized water and polyethylene glycol is added in 25 mass ratioes（Go from
The volume ratio of sub- water and polyethylene glycol is 1：0.2）Slurry is obtained after stirring evenly, and the lactitol and 0.8% for accounting for slurry gross weight 2% is added
Citric acid, after mixing, stand 15min, then be added 1mol/L NaOH solution tune pH value to 11.0,180 W ultrasound
Then 2.5 h, centrifuging and taking supernatant use the HCl solution of 1mol/L to adjust pH to 3.80, centrifugation goes supernatant that precipitation, precipitation is stayed to set
It is lyophilized in vacuum freeze drier, obtains -20 DEG C of freeze-dried powder and store for future use；
（3）It weighs freeze-dried powder 7g and is dissolved in 1000mL deionized waters, 0.56g beeswax powders are added, is placed in 80 DEG C of water-baths and preheats 20
Min obtains mixed liquor；
（4）It is 8.0 that mixed liquor is adjusted pH using the NaOH solution of 1mol/L, and the basic protein for accounting for mixed liquor weight 2.5% is added
Enzyme and flavor protease（Ratio is same as above）, digest 2h in 56 DEG C of thermostat water bath, in enzymolysis process, made using NaOH solution
PH of mixed remains unchanged, and enzymolysis liquid is placed in boiling water after enzymolysis and heats 10min enzyme deactivations work, and ice bath cools down rapidly, from
The heart collects supernatant freeze-drying.
Comparative example 1
（1）Microwave thawing after the peony seeds dregs of rice are freezed at -20 DEG C, it is spare to obtain peony seeds dregs of rice powder at powder for drying and grinding after degreasing；
（2）By peony seeds dregs of rice powder according to volume ratio 1：Deionized water is added in 25 mass ratioes, and slurry is obtained after stirring evenly, and addition accounts for slurry
Expect the lactitol of gross weight 1.5% and 1% citric acid, after mixing, stands 15min, the NaOH solution of 1mol/L is then added
Adjust pH value to 11.0,180 W, 2.5 h of ultrasound, then centrifuging and taking supernatant uses the HCl solution adjusting pH to 3.80 of 1mol/L, from
The heart goes supernatant to stay precipitation, and precipitation, which is placed in vacuum freeze drier, to be lyophilized, and is obtained -20 DEG C of freeze-dried powder and is stored for future use；
（3）It weighs freeze-dried powder 7g and is dissolved in 1000mL deionized waters, 0.35g beeswax powders are added, is placed in 80 DEG C of water-baths and preheats 20
Min obtains mixed liquor；
（4）It is 8.0 that mixed liquor is adjusted pH using the NaOH solution of 1mol/L, and the basic protein for accounting for mixed liquor weight 2.5% is added
Enzyme and flavor protease, digest 2h in 56 DEG C of thermostat water bath, in enzymolysis process, so that pH of mixed is protected using NaOH solution
It holds constant, enzymolysis liquid is placed in boiling water after enzymolysis and heats 10min enzyme deactivations and lives, ice bath cools down rapidly, centrifuges, and collects supernatant
Liquid is freeze-dried.
Comparative example 2
（1）Microwave thawing after the peony seeds dregs of rice are freezed at -20 DEG C, it is spare to obtain peony seeds dregs of rice powder at powder for drying and grinding after degreasing；
（2）By peony seeds dregs of rice powder according to volume ratio 1：The mixed solution of deionized water and polyethylene glycol is added in 25 mass ratioes（Go from
The volume ratio of sub- water and polyethylene glycol is 1：0.1）Slurry is obtained after stirring evenly, and after mixing, is stood 15-20min, is then added
Enter the NaOH solution tune pH value of 1mol/L to 11.0,180 W, 2.5 h of ultrasound, then centrifuging and taking supernatant uses the HCl of 1mol/L
Solution adjusts pH to 3.80, and centrifugation goes supernatant to stay precipitation, and precipitation, which is placed in vacuum freeze drier, to be lyophilized, and -20 DEG C of freeze-dried powder is obtained
It stores for future use；
（3）It weighs freeze-dried powder 7g and is dissolved in 1000mL deionized waters, 0.35g beeswax powders are added, is placed in 80 DEG C of water-baths and preheats 20
Min obtains mixed liquor；
（4）It is 8.0 that mixed liquor is adjusted pH using the NaOH solution of 1mol/L, and the basic protein for accounting for mixed liquor weight 2.5% is added
Enzyme and flavor protease, digest 2h in 56 DEG C of thermostat water bath, in enzymolysis process, so that pH of mixed is protected using NaOH solution
It holds constant, enzymolysis liquid is placed in boiling water after enzymolysis and heats 10min enzyme deactivations and lives, ice bath cools down rapidly, centrifuges, and collects supernatant
Liquid is freeze-dried.
Comparative example 3
（1）Microwave thawing after the peony seeds dregs of rice are freezed at -20 DEG C, it is spare to obtain peony seeds dregs of rice powder at powder for drying and grinding after degreasing；
（2）By peony seeds dregs of rice powder according to volume ratio 1：Deionized water is added in 25 mass ratioes, and slurry is obtained after stirring evenly, is then added
The NaOH solution tune pH value of 1mol/L is to 11.0,180 W, 2.5 h of ultrasound, centrifuging and taking supernatant, then uses the HCl of 1mol/L molten
Liquid adjusts pH to 3.80, and centrifugation goes supernatant to stay precipitation, and precipitation, which is placed in vacuum freeze drier, to be lyophilized, and obtains -20 DEG C of storages of freeze-dried powder
It deposits spare；
（3）It weighs freeze-dried powder 7g and is dissolved in 1000mL deionized waters, be placed in 80 DEG C of water-baths and preheat 20 min, obtain mixed liquor；
（4）It is 8.0 that mixed liquor is adjusted pH using the NaOH solution of 1mol/L, and the basic protein for accounting for mixed liquor weight 2.5% is added
Enzyme and flavor protease, digest 2h in 56 DEG C of thermostat water bath, in enzymolysis process, so that pH of mixed is protected using NaOH solution
It holds constant, enzymolysis liquid is placed in boiling water after enzymolysis and heats 10min enzyme deactivations and lives, ice bath cools down rapidly, centrifuges, and collects supernatant
Liquid is freeze-dried.
Comparative example 4
（1）Microwave thawing after the peony seeds dregs of rice are freezed at -20 DEG C, it is spare to obtain peony seeds dregs of rice powder at powder for drying and grinding after degreasing；
（2）By peony seeds dregs of rice powder according to volume ratio 1：Deionized water is added in 25 mass ratioes, and slurry is obtained after stirring evenly, and addition accounts for slurry
Expect the citric acid of gross weight 2.5%, after mixing, stands 15-20min, the NaOH solution tune pH value that 1mol/L is then added arrives
Then 11.0,180 W, 2.5 h of ultrasound, centrifuging and taking supernatant use the HCl solution of 1mol/L to adjust pH to 3.80, supernatant is removed in centrifugation
Precipitation is stayed, precipitation, which is placed in vacuum freeze drier, to be lyophilized, and is obtained -20 DEG C of freeze-dried powder and is stored for future use；
（3）It weighs freeze-dried powder 7g and is dissolved in 1000mL deionized waters, be placed in 80 DEG C of water-baths and preheat 20 min, obtain mixed liquor；
（4）It is 8.0 that mixed liquor is adjusted pH using the NaOH solution of 1mol/L, and the basic protein for accounting for mixed liquor weight 2.5% is added
Enzyme and flavor protease, digest 2h in 56 DEG C of thermostat water bath, in enzymolysis process, so that pH of mixed is protected using NaOH solution
It holds constant, enzymolysis liquid is placed in boiling water after enzymolysis and heats 10min enzyme deactivations and lives, ice bath cools down rapidly, centrifuges, and collects supernatant
Liquid is freeze-dried.
（One）By the purity of degree of hydrolysis and the product finally prepared in embodiment 1-2 and comparative example 1-4 preparation process
It is detected：
（1）Degree of hydrolysis measures
PH-stat method of the measurement of degree of hydrolysis with reference to foundation such as Adler-Nissen.
In formula：DH-degree of hydrolysis；NbThe concentration of-NaOH（mol/L）；MpThe quality of-aminosal（g）；The body of B-NaOH
Product（mL）；α-average dissociation degree；htotMM number of peptide bond in-every gram material protein, peony seeds albumen be h=
7.84mmol/g；
Concrete outcome is shown in Table 1.
Table 1
（Two）Embodiment 1-2 and comparative example the 1-4 anti-oxidation peptide prepared are subjected to Scavenging action to hydroxyl free radical and pyrogallol oxygen
The inhibiting rate for the property changed is detected, and concrete outcome is shown in Table 2.
（Three）Embodiment 1-2 and comparative example 1-4 are in carrying out preparation extraction process, the side of 1-2 offers of the embodiment of the present invention
Method, enzymolysis liquid show the distinctive fragrance of the peony seeds dregs of rice and normal fermented flavour, and in comparative example 1-4 fermentation process, Yi Fa
The matter that changes tapinoma-odour, Partial fermentation tank is rotten can not to be normally carried out experiment, and comparative example during the experiment, is also easy to produce miscellaneous bacteria and hair
Raw lamination.
（Four）The screening of protease in enzymolysis process
By 5 parts of the mixed liquor of preparation, it is separately added into 5 kinds of enzymes（Alkali protease, neutral proteinase, compound protease, flavor albumen
Enzyme, papain）, enzyme concentration 45mg digests 8 h under the conditions of respective optimum temperature and pH, later, enzymolysis liquid is placed in boiling
10 min enzyme deactivations are lived in water, and centrifugation takes supernatant to measure degree of hydrolysis and inoxidizability, filters out most suitable peony seeds proteolysis
Protease, be specifically shown in Fig. 1, Fig. 1 is different protease hydrolyzeds in peony seeds protein antioxidant peptide preparation process of the present invention
Hydrolysis curves figure.In method provided by the invention, when alkali protease or flavor protease is only added, alkali protease
Addition need to add to 4.6%, and when flavor protease is only added, effect is poor, after the two is used in compounding, Neng Gouxian
Write the usage amount for reducing protease.
Claims (9)
1. a kind of method for extracting anti-oxidation peptide in dregs of rice from peony seeds, which is characterized in that include the following steps：
（1）Microwave thawing after the peony seeds dregs of rice are freezed, it is spare to obtain peony seeds dregs of rice powder at powder for drying and grinding after degreasing；
（2）By peony seeds dregs of rice powder according to volume ratio 1：Solvent is added in 25 mass ratioes, and lactitol and citric acid, mixing are added in slurry
After uniformly, 15-20min is stood, then addition NaOH solution tune pH value to 11.0,180 W, 2.5 h of ultrasound, centrifuging and taking supernatant,
Then pH to 3.80 is adjusted with HCl solution, centrifugation goes supernatant to stay precipitation, and precipitation, which is placed in vacuum freeze drier, to be lyophilized, and must be frozen
- 20 DEG C of dry powder stores for future use；
（3）It weighs freeze-dried powder and is dissolved in deionized water, beeswax is added, is placed in 80 DEG C of water-baths and preheats 20 min, obtain mixed liquor；
（4）It is 8.0 that mixed liquor, which is adjusted pH, and alkali protease and flavor protease is added, then enzymolysis liquid is placed in boiling by enzymolysis
It heats 10min enzyme deactivations in water to live, ice bath cools down rapidly, centrifuges, and collects supernatant freeze-drying.
2. according to the method described in claim 1, it is characterized in that, step（1）In, the peony seeds dregs of rice are freezed at -20 DEG C, so
Microwave thawing afterwards.
3. according to the method described in claim 1, it is characterized in that, the solvent is molten for the mixing of deionized water and polyethylene glycol
Liquid；The deionized water and the volume ratio of polyethylene glycol are 1：0.1-0.2.
4. method according to claim 1 or 3, which is characterized in that the addition of the lactitol accounts for the 1- of slurry gross weight
2%；The addition of the citric acid accounts for the 0.8-1% of slurry gross weight.
5. according to the method described in claim 4, it is characterized in that, the NaOH solution and the concentration of HCl solution are 1mol/
L。
6. according to the method described in claim 1, it is characterized in that, step（3）In, it is added in the freeze-dried powder per 0.7g
100mL deionized waters.
7. method according to claim 1 or 6, which is characterized in that the addition of the beeswax accounts for freeze-dried powder weight
0.05-0.08 times.
8. according to the method described in claim 1, it is characterized in that, the addition of the alkali protease and flavor protease is
The 2.5% of mixed liquor weight；The mass ratio of the alkali protease and flavor protease is 1.5：1.
9. according to the method described in claim 8, it is characterized in that, the temperature of the enzymolysis be 56 DEG C, enzymolysis time 2h.
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| CN116789736A (en) * | 2023-04-27 | 2023-09-22 | 山东牡丹爱生物科技有限公司 | Active peony peptide and application thereof as antioxidant and antifatigue preparation |
| CN116478261B (en) * | 2023-04-27 | 2023-10-27 | 德州蓝力生物技术有限公司 | Anti-fatigue antioxidant peony peptide and preparation method thereof |
| CN116970673A (en) * | 2023-09-11 | 2023-10-31 | 山东大树达孚特膳食品有限公司 | Anti-fatigue peony peptide and preparation method thereof |
| CN116970673B (en) * | 2023-09-11 | 2024-02-23 | 山东大树达孚特膳食品有限公司 | Anti-fatigue peony peptide and preparation method thereof |
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