CN106801079A - The method that a kind of pair of enzyme stepwise discretization Carapax Eriocheir sinensis prepare antioxidation active peptides - Google Patents
The method that a kind of pair of enzyme stepwise discretization Carapax Eriocheir sinensis prepare antioxidation active peptides Download PDFInfo
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- CN106801079A CN106801079A CN201611186984.2A CN201611186984A CN106801079A CN 106801079 A CN106801079 A CN 106801079A CN 201611186984 A CN201611186984 A CN 201611186984A CN 106801079 A CN106801079 A CN 106801079A
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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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- 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
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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
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
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Abstract
The invention discloses the method that a kind of pair of enzyme stepwise discretization Carapax Eriocheir sinensis prepare antioxidation active peptides, comprise the following steps：(1) pretreatment of raw material；(2) double enzyme enzymolysis (3) centrifugations；(4) UF membrane；(5) vacuum freeze drying.The present invention is with crab shell as raw material, crab shell obtains the crab shell anti-oxidation peptide of solid through double enzyme stepwise discretizations, centrifugation, UF membrane, vacuum freeze drying after crushing, processing step is simple, process stabilization is controllable, energy-conservation, efficient, environmental protection, the comprehensive utilization of crab processing waste resource is realized, its application value is improved, the economic worth of crab is effectively increased.
Description
Technical field
The present invention relates to biologically active peptide preparing technical field, prepared more particularly, to a kind of pair of enzyme stepwise discretization Carapax Eriocheir sinensis
The method of antioxidation active peptides.
Background technology
Ocean crustacean aboundresources, is counted according to FAO fishery portion of the World Food Programme, and world ocean crust is moved within 1996
Thing total output reaches 510.7 ten thousand tons, and wherein crab class accounts for the 23.88% of total output.Substantial amounts of leftover bits and pieces can be produced after crab class is processed
Material, such as crab pin, crab shell on the back, due to the aspect reason such as technology and understanding, most of these shrimp and crab shells are often arbitrarily abandoned, and are made
Into great pollution and the wasting of resources.It is existing to add and domestic production enterprise is still in the junior stage to crab shell processing and utilization
In addition to small part coarse crushing processes raw material as fish meal, majority be used to produce chitin work Application way, and chitin is given birth to
Enterprise is produced to face environmental protection pressure mostly and stop production.Therefore how this resource is maximally utilized, is developed new product
Aquatic Product Process Industry urgent problem is turned into.
In recent years, the research for being comprehensively utilized to disposal from fishery product processing mainly uses protease to carry gradually into focus
Protolysate is taken, for the exploitation of flavoring or food additives.Rich in protein is rich in shrimp and crab shells, and it is numerous right
The trace element such as the beneficial nutrient of human body and iron, zinc, copper, using biological enzymolysis technology, crab shell resource is effectively opened
Hair is utilized, and preparing to turn into has anti-oxidation function, and nutritious product, will greatly improve its application value, is effectively increased
The economic worth of crab.
The content of the invention
Simple the invention aims to provide a kind of processing step, process stabilization is controllable, energy-conservation, efficient, green ring
The method that the double enzyme stepwise discretization Carapax Eriocheir sinensis protected prepare antioxidation active peptides, anti-oxidant titanium is prepared using crab shell, realizes that crab adds
The comprehensive utilization of work waste material, improves its application value, effectively increases the economic worth of crab.
To achieve these goals, the present invention uses following technical scheme：
The method that a kind of pair of enzyme stepwise discretization Carapax Eriocheir sinensis prepare antioxidation active peptides, comprises the following steps：
(1) pretreatment of raw material：Crab shell is washed, is dried, size-reduced machine is crushed, and obtains crab shell powder, stand-by.
(2) double enzyme enzymolysis：By crab shell powder and water in mass ratio 1：(5~10) after mixing, regulation pH is 2.0~3.0,30
~40 DEG C of 10~20min of insulation, are incorporated as the acid protease of crab shell powder quality 0.1~0.3%, and insulation 6~8h of enzymolysis rises to
95~100 DEG C of 15~20min of enzyme that go out, are cooled to 50~60 DEG C, and pH is to 6.5~7.5 for regulation, be incorporated as crab shell powder quality 0.1~
0.3% flavor protease, in 50~60 DEG C of insulation 2~4h of enzymolysis, is warming up to 95~100 DEG C of 10~20min of enzyme that go out, and must digest
Liquid.Key point of the invention is enzymolysis process, and different protease is different to the action site of protein, single enzymolysis effect
Site is more single, causes the degree of hydrolysis of protein relatively low, and the present invention uses double enzyme resolving tech, due to acid protease and local flavor
The action site of protease differs greatly, therefore can effectively improve the degree of hydrolysis of crab shell albumen, so as to obtain the work of small molecule
Property peptide.Described acid protease be by after aspergillus niger cultivation and fermentation using modern biotechnology refine it is safe efficient
Biological food level enzyme preparation, its molecular weight 35kDa or so, enzyme activity is 20000~50000U/g；Described flavor protease is
Fermented by aspergillus oryzae strain, purified, compounding food-grade compound fertilizer production, enzyme activity is >=30000U/g.
(3) centrifugation：Enzymolysis liquid is centrifuged at a high speed, supernatant liquor is taken, anti-oxidation peptide crude extract is obtained.
(4) UF membrane：Anti-oxidation peptide crude extract is carried out into separating-purifying using milipore filter, crab shell anti-oxidation peptide is obtained molten
Liquid.UF membrane removal is further improving the pure of crab shell anti-oxidation peptide without the relatively low composition of antioxidation activity or antioxidation activity
While spending, the influence of structure and activity to anti-oxidation peptide is smaller.
(5) vacuum freeze drying：By anti-oxidation peptide solution vacuum freeze drying, the crab shell antioxidation activity of solid is obtained final product
Peptide.
Preferably, in step (2), ultracentrifugal technological parameter is：6000~10000rpm/min of centrifugal rotational speed, when
Between 10~30min.
Preferably, in step (2), the acid protease enzyme activity is 20000~50000U/g, the local flavor albumen
Enzyme enzyme activity >=30000U/g.
Preferably, in step (2), enzymolysis process is carried out under agitation.
Therefore, the present invention has the advantages that：With crab shell as raw material, crab shell passes through double enzyme substep enzymes to the present invention after crushing
Solution, centrifugation, UF membrane, vacuum freeze drying obtain the crab shell anti-oxidation peptide of solid, and processing step is simple, and process stabilization can
Control, energy-conservation, efficient, environmental protection, realizes the comprehensive utilization of crab processing waste resource, improves its application value, effectively
Increased the economic worth of crab.
Specific embodiment
Below by specific embodiment, the present invention will be further described.
Embodiment 1
(1) pretreatment of raw material：Crab shell is washed, is dried, size-reduced machine is crushed, and obtains crab shell powder, stand-by；
(2) double enzyme enzymolysis：By crab shell powder and water in mass ratio 1：After 5 mixing, regulation pH is 2.0, in 30 DEG C of insulations
10min, is incorporated as the acid protease that crab shell powder quality 0.1%, enzyme activity is 20000U/g, under agitation insulation enzymolysis
6h, rises to 95 DEG C of enzyme 15min that go out, and is cooled to 50 DEG C, and pH is to 6.5 for regulation, be incorporated as crab shell powder quality 0.1%, enzyme activity >=
The flavor protease of 30000U/g, under agitation in 50 DEG C of insulation enzymolysis 2h, is warming up to 95 DEG C of enzyme 10min that go out, and must digest
Liquid；
(3) centrifugation：Enzymolysis liquid is centrifuged at a high speed, supernatant liquor is taken, anti-oxidation peptide crude extract is obtained, at a high speed
The technological parameter of centrifugation is：Centrifugal rotational speed 6000rpm/min, time 10min；
(4) UF membrane：Anti-oxidation peptide crude extract is carried out into separating-purifying using milipore filter, crab shell anti-oxidation peptide is obtained molten
Liquid；
(5) vacuum freeze drying：By anti-oxidation peptide solution vacuum freeze drying, the crab shell antioxidation activity of solid is obtained final product
Peptide.
The crab shell anti-oxidation peptide that will be obtained represents through Antioxidative Activity Determination with ascorbic acid, crab shell anti-oxidation peptide
Elimination effect to DPPH is 10.5mg AAE/g samples, and the yield of anti-oxidation peptide is 240mg/g raw materials；Sensory evaluation is 4.
Embodiment 2
(1) pretreatment of raw material：Crab shell is washed, is dried, size-reduced machine is crushed, and obtains crab shell powder, stand-by；
(2) double enzyme enzymolysis：By crab shell powder and water in mass ratio 1：After 10 mixing, regulation pH is 3.0, in 40 DEG C of insulations
20min, is incorporated as the acid protease that crab shell powder quality 0.3%, enzyme activity is 50000U/g, under agitation insulation enzymolysis
8h, rises to 100 DEG C of enzyme 20min that go out, and is cooled to 60 DEG C, and pH is to 7.5 for regulation, be incorporated as crab shell powder quality 0.3%, enzyme activity >=
The flavor protease of 30000U/g, under agitation in 60 DEG C of insulation enzymolysis 4h, is warming up to 100 DEG C of enzyme 20min that go out, and must digest
Liquid；
(3) centrifugation：Enzymolysis liquid is centrifuged at a high speed, supernatant liquor is taken, anti-oxidation peptide crude extract is obtained, at a high speed
The technological parameter of centrifugation is：Centrifugal rotational speed 10000rpm/min, time 30min；
(4) UF membrane：Anti-oxidation peptide crude extract is carried out into separating-purifying using milipore filter, crab shell anti-oxidation peptide is obtained molten
Liquid；
(5) vacuum freeze drying：By anti-oxidation peptide solution vacuum freeze drying, the crab shell antioxidation activity of solid is obtained final product
Peptide.
The crab shell anti-oxidation peptide that will be obtained represents through Antioxidative Activity Determination with ascorbic acid, crab shell anti-oxidation peptide
Elimination effect to DPPH is 10.2mg AAE/g samples, and the yield of anti-oxidation peptide is 180mg/g raw materials；Sensory evaluation is 4.
Embodiment 3
(1) pretreatment of raw material：Crab shell is washed, is dried, size-reduced machine is crushed, and obtains crab shell powder, stand-by；
(2) double enzyme enzymolysis：By crab shell powder and water in mass ratio 1：After 7 mixing, regulation pH is 2.5, in 35 DEG C of insulations
15min, is incorporated as the acid protease that crab shell powder quality 0.2%, enzyme activity is 30000U/g, under agitation insulation enzymolysis
7h, rises to 98 DEG C of enzyme 17min that go out, and is cooled to 55 DEG C, and pH is to 7.2 for regulation, be incorporated as crab shell powder quality 0.2%, enzyme activity >=
The flavor protease of 30000U/g, under agitation in 55 DEG C of insulation enzymolysis 3h, is warming up to 97 DEG C of enzyme 15min that go out, and must digest
Liquid；
(3) centrifugation：Enzymolysis liquid is centrifuged at a high speed, supernatant liquor is taken, anti-oxidation peptide crude extract is obtained, at a high speed
The technological parameter of centrifugation is：Centrifugal rotational speed 8000rpm/min, time 20min；
(4) UF membrane：Anti-oxidation peptide crude extract is carried out into separating-purifying using milipore filter, crab shell anti-oxidation peptide is obtained molten
Liquid；
(5) vacuum freeze drying：By anti-oxidation peptide solution vacuum freeze drying, the crab shell antioxidation activity of solid is obtained final product
Peptide.
The crab shell anti-oxidation peptide that will be obtained represents through Antioxidative Activity Determination with ascorbic acid, crab shell anti-oxidation peptide
Elimination effect to DPPH is 9.8mg AAE/g samples, and the yield of anti-oxidation peptide is 249mg/g raw materials；Sensory evaluation is 4.
DPPH clearance rate assay methods used by the present invention are concretely comprised the following steps：
The crab shell anti-oxidation peptide solution of 1.5mL suitably dilutions is taken, (95% ethanol is molten to add 3.0mL 0.09mg/mL DPPH
Liquid), room temperature avoid light place 5min is well mixed, managed by zeroing of 95% ethanol, determination sample light absorption value at 517nm.Wherein
Blank with 95% ethanol solution substitute sample, standard items be ascorbic acid (AAE) solution, concentration be 0.01,0.02,
0.03、0.04mg/mL。
Result represents with every g crab shells anti-oxidation peptide equivalent to the mg numbers of ascorbic acid, i.e. mg AAE/g samples.
The sensory evaluation method that uses of the present invention for：As shown in 8 trained sensory evaluation group members according to table 1
Standards of grading, sensory evaluation scores are carried out to crab shell anti-oxidation peptide.
The sensory evaluation standards of grading of table 1
Embodiment described above is a kind of preferably scheme of the invention, not makees any formal to the present invention
Limitation, also has other variants and remodeling on the premise of without departing from the technical scheme described in claim.
Claims (4)
1. the method that a kind of pair of enzyme stepwise discretization Carapax Eriocheir sinensis prepare antioxidation active peptides, it is characterised in that comprise the following steps：
(1) pretreatment of raw material：Crab shell is washed, is dried, size-reduced machine is crushed, and obtains crab shell powder, stand-by；
(2) double enzyme enzymolysis：By crab shell powder and water in mass ratio 1：(5~10) after mixing, regulation pH is 2.0~3.0,30~40
DEG C 10~20min of insulation, is incorporated as the acid protease of crab shell powder quality 0.1~0.3%, insulation 6~8h of enzymolysis, rise to 95~
100 DEG C of 15~20min of enzyme that go out, are cooled to 50~60 DEG C, and pH is to 6.5~7.5 for regulation, is incorporated as crab shell powder quality 0.1~0.3%
Flavor protease, in 50~60 DEG C insulation enzymolysis 2~4h, be warming up to 95~100 DEG C of 10~20min of enzyme that go out, obtain enzymolysis liquid；
(3) centrifugation：Enzymolysis liquid is centrifuged at a high speed, supernatant liquor is taken, anti-oxidation peptide crude extract is obtained；
(4) UF membrane：Anti-oxidation peptide crude extract is carried out into separating-purifying using milipore filter, crab shell anti-oxidation peptide solution is obtained；
(5) vacuum freeze drying：By anti-oxidation peptide solution vacuum freeze drying, the crab shell antioxidation active peptides of solid are obtained final product.
2. the method that a kind of pair of enzyme stepwise discretization Carapax Eriocheir sinensis according to claim 1 prepare antioxidation active peptides, its feature
It is that in step (2), ultracentrifugal technological parameter is：6000~10000rpm/min of centrifugal rotational speed, 10~30min of time.
3. the method that a kind of pair of enzyme stepwise discretization Carapax Eriocheir sinensis according to claim 1 and 2 prepare antioxidation active peptides, it is special
Levy and be, in step (2), the acid protease enzyme activity be 20000~50000U/g, the flavor protease enzyme activity >=
30000U/g。
4. the method that a kind of pair of enzyme stepwise discretization Carapax Eriocheir sinensis according to claim 1 prepare antioxidation active peptides, its feature
It is that in step (2), enzymolysis process is carried out under agitation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107400158A (en) * | 2017-09-28 | 2017-11-28 | 南京财经大学 | A kind of method that stepwise discretization prepares high yield anti-inflammatory rapeseed peptide (rsp) |
| CN107751946A (en) * | 2017-09-22 | 2018-03-06 | 浙江海洋大学 | A kind of method that seafood condiment is prepared using crab shell |
| CN108530208A (en) * | 2018-07-03 | 2018-09-14 | 合肥百绿盛农业科技有限公司 | It is a kind of using waste as soil activating agent of raw material and preparation method thereof |
| CN109222043A (en) * | 2018-08-27 | 2019-01-18 | 合肥工业大学 | A method of seasoning is made using shrimp and crab shells |
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| CN104878061A (en) * | 2015-05-15 | 2015-09-02 | 海南大学 | Double-enzyme preparation method of oyster meat nutrient solution with antioxidation function |
| CN105175495A (en) * | 2015-07-09 | 2015-12-23 | 浙江海洋学院 | Use of degreased crab shell antioxidant peptide |
| CN105198962A (en) * | 2015-07-09 | 2015-12-30 | 浙江海洋学院 | Defatted crab shell antioxidative polypeptide |
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2016
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|---|---|---|---|---|
| CN104878061A (en) * | 2015-05-15 | 2015-09-02 | 海南大学 | Double-enzyme preparation method of oyster meat nutrient solution with antioxidation function |
| CN105175495A (en) * | 2015-07-09 | 2015-12-23 | 浙江海洋学院 | Use of degreased crab shell antioxidant peptide |
| CN105198962A (en) * | 2015-07-09 | 2015-12-30 | 浙江海洋学院 | Defatted crab shell antioxidative polypeptide |
Non-Patent Citations (1)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107751946A (en) * | 2017-09-22 | 2018-03-06 | 浙江海洋大学 | A kind of method that seafood condiment is prepared using crab shell |
| CN107400158A (en) * | 2017-09-28 | 2017-11-28 | 南京财经大学 | A kind of method that stepwise discretization prepares high yield anti-inflammatory rapeseed peptide (rsp) |
| CN108530208A (en) * | 2018-07-03 | 2018-09-14 | 合肥百绿盛农业科技有限公司 | It is a kind of using waste as soil activating agent of raw material and preparation method thereof |
| CN109222043A (en) * | 2018-08-27 | 2019-01-18 | 合肥工业大学 | A method of seasoning is made using shrimp and crab shells |
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Application publication date: 20170606 |