CN106701874A - Preparation method for phycocyanin polypeptide - Google Patents
Preparation method for phycocyanin polypeptide Download PDFInfo
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- CN106701874A CN106701874A CN201611018464.0A CN201611018464A CN106701874A CN 106701874 A CN106701874 A CN 106701874A CN 201611018464 A CN201611018464 A CN 201611018464A CN 106701874 A CN106701874 A CN 106701874A
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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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Abstract
The invention relates to a preparation method for a phycocyanin polypeptide, which includes the following steps: phycocyanin is added with water for being stirred to be sufficiently dissolved, so that a phycocyanin solution is obtained; the phycocyanin solution is added with papain or neutral protease, uniform stirring is carried out and then ultrasonical treatment is carried out for 30 to 50 minutes, so that an enzymolysis product is obtained; the enzymolysis product is heated to 85 DEG C to 95 DEG C which is kept for 5 to 15 minutes, and after enzyme deactivation, cooling and centrifugation, supernatant, i.e. enzymolysis liquid, is extracted; ultrafiltration treatment is carried out on the enzymolysis liquid, so that an ultrafiltrate is obtained; the ultrafiltrate is concentrated under vacuum, and after spray-drying treatment, the phycocyanin polypeptide is obtained. The preparation method for the phycocyanin polypeptide adopts ultrasound-assisted enzymolysis, ultrasonic waves have a cavitation effect, produced air bubbles are squeezed to break to instantly produce extremely strong mechanical shear force, so that protein is degraded or protein conformation is changed, consequently, more hydrophilic groups are promoted to expose, the solubility of phycocyanin in water is increased, the binding between enzyme and phycocyanin substrate is benefited, and the efficiency of enzymolysis of phycocyanin can be remarkably increased.
Description
Technical field
The present invention relates to protein digestion technical field, more particularly to a kind of preparation method of phycocyanin polypeptide.
Background technology
Phycocyanin (Phycocyanin, also known as PC) is a kind of photosynthetic complementary colors being present in red algae and Cells of Blue-green Algae
Element, can efficient capture luminous energy.Research in recent years finds that phycocyanin has anti-oxidant, anticancer, anti-inflammatory and neuroprotection etc. many
Bioactivity is planted, the focus of research natural marine medicine is had become to its research.Research of the China to phycocyanin is started from
20 century 70s, have passed through the research and development over more than 30 years, although have larger progress, but big all in laboratory water
It is flat.Meanwhile, functional food based on phycocyanin is few, in terms of the research and development of medicine or blank.Enzyme hydrolysis can
To improve the functional characteristic of protein, the peptide for obtaining has the incomparable physicochemical characteristics of some protein.If using
Phycocyanin is degraded to soluble oligopeptides by the method for enzymolysis, then be greatly improved its physicochemical property in food, and may
Obtain its precursor without physiologically active.Patent is had at present is related to the enzymolysis of phycocyanin.Patent spirulina phycocyanin
Enzymolysis product and its application (application number 200910178865.6) provide spirulina phycocyanin enzymolysis product, including spiral
The functional food of the enzymolysis product of algae phycocyanin and the enzymolysis product comprising spirulina phycocyanin and pharmaceutically acceptable
Carrier pharmaceutical composition.A kind of utilization phycocyanin of patent prepares the method (application number of Caspase-3 activation
201110377690.6) disclose and phycocyanin is extracted from spirulina powder with for one kind, using trypsin hydrolysis algae
Azurin, the isolated activation than phycocyanin to Caspase-3 enzyme activities in itself is more effective, to normal cytotoxic
The method of the peptide of activity.But existing phycocyanin enzyme solution, single zymolysis technique is typically with to phycocyanin
Digested, haveed the shortcomings that proteolytic efficiency is low.
The content of the invention
In consideration of it, being necessary to provide a kind of preparation method of proteolytic efficiency phycocyanin polypeptide higher.
A kind of preparation method of phycocyanin polypeptide, comprises the following steps：
Add water in phycocyanin, stirring makes it fully dissolve, and obtains phycocyanin solution；
To addition papain or neutral proteinase in the phycocyanin solution, after stirring, ultrasonically treated 30
~50min, obtains enzymolysis product；
The enzymolysis product is warming up to 85 DEG C~95 DEG C and 5~15min is incubated, 30 DEG C~40 DEG C is cooled to after the enzyme that goes out,
Centrifugation, takes supernatant, as enzymolysis liquid；
The enzymolysis liquid is carried out into hyperfiltration treatment, ultrafiltrate is obtained；
The ultrafiltrate is concentrated in vacuo, spray drying treatment obtains the phycocyanin polypeptide.
Wherein in one embodiment, add water in phycocyanin, stirring makes it fully dissolve, and obtains phycocyanin molten
In the operation of liquid, the solid-liquid ratio of phycocyanin and water is 1:10~1:50.
Wherein in one embodiment, add water in phycocyanin, stirring makes it fully dissolve, and obtains phycocyanin molten
In the operation of liquid, using temperature constant magnetic stirring, temperature is 50 DEG C~55 DEG C.
Wherein in one embodiment, the mass ratio of papain and phycocyanin is 1:100~1:20.
Wherein in one embodiment, the mass ratio of neutral proteinase and phycocyanin is 1:30~1:20.
Wherein in one embodiment, before papain is added, also including by the pH of the phycocyanin solution
The step of regulation is to 5.0~7.0.
Wherein in one embodiment, before neutral proteinase is added, also including by the pH of the phycocyanin solution
The step of regulation is to 6.0~7.0.
Wherein in one embodiment, add papain carry out it is ultrasonically treated during, reaction temperature be 50 DEG C
~60 DEG C.
Wherein in one embodiment, add neutral proteinase carry out it is ultrasonically treated during, reaction temperature be 45 DEG C
~55 DEG C.
Wherein in one embodiment, in ultrasonically treated operation, ultrasonic power is 300~800W.
The preparation method of above-mentioned phycocyanin polypeptide, is digested using ultrasonic wave added, and ultrasonic wave has cavitation, generation
Bubble ruptures through extruding, can produce extremely strong mechanical shear stress with moment, makes protein degradation or changes protein conformation, promotees
Its hydrophilic radical is more exposed, improve solubility of the phycocyanin in water, be conducive to enzyme and phycocyanin substrate
With reference to can within a short period of time significantly improve the enzymolysis efficiency of phycocyanin.
Brief description of the drawings
Fig. 1 is the flow chart of the preparation method of the phycocyanin polypeptide of an implementation method；
Fig. 2 is removing influence figure of the phycocyanin peptide concentration for preparing of embodiment 1 on DPPH free radicals.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated, it will be appreciated that instantiation described herein only to explain the present invention, is not used to limit this
Invention.
Fig. 1 is refer to, the preparation method of the phycocyanin polypeptide of an implementation method comprises the following steps：
S10, add water to phycocyanin, stirring makes it fully dissolve, obtain phycocyanin solution.
Wherein, the purity A of phycocyanin620/A280>2.The solid-liquid ratio of phycocyanin and water can be 1:10~1:50.
In the operation of S10, phycocyanin is dissolved using temperature constant magnetic stirring, temperature can be 50 DEG C~55 DEG C.Stirring
Time can be 20~40min.
S20, in phycocyanin solution add papain or neutral proteinase, after stirring, ultrasonically treated 30
~50min, obtains enzymolysis product.
Wherein, it is ultrasonically treated to be carried out in water bath sonicator device.
Wherein, the enzyme activity of papain can be 5 × 105~10 × 105U/g.Papain and phycocyanin
Mass ratio can be 1:100~1:20.Add papain carry out it is ultrasonically treated during, reaction temperature can be 50 DEG C
~60 DEG C.Ultrasonic power can be 300~800W.Before papain is added, can also include phycocyanin solution
PH the step of adjust to 5.0~7.0.Further, the pH regulations of phycocyanin solution can be carried out using 1M hydrochloric acid.
Wherein, the enzyme activity of neutral proteinase is 1 × 105~3 × 105U/g.The quality of neutral proteinase and phycocyanin
Than that can be 1:30~1:20.Add neutral proteinase carry out it is ultrasonically treated during, reaction temperature can be 45 DEG C~55
℃.Ultrasonic power can be 300~800W.Before neutral proteinase is added, can also include the pH of phycocyanin solution
The step of regulation is to 6.0~7.0.Further, the pH regulations of phycocyanin solution can be carried out using 1M hydrochloric acid.
S30, enzymolysis product is warming up to 85 DEG C~95 DEG C and be incubated 5~15min, after the enzyme that goes out cool down, centrifugation, take supernatant,
As enzymolysis liquid.
Wherein, heating rate can be 20~30 DEG C/min.
In S30, after the enzyme that goes out, it is centrifuged again after being cooled to 30 DEG C~40 DEG C.
In centrifugally operated, can be using the rotating speed of 10000r/min centrifugation 10min.
S40, enzymolysis liquid is carried out into hyperfiltration treatment, obtain ultrafiltrate.
Wherein it is possible to use the trapped molecular weight to carry out hyperfiltration treatment for 1kD~10kD milipore filters.
S50, ultrafiltrate is concentrated in vacuo, spray drying treatment obtains phycocyanin polypeptide.
The preparation method of above-mentioned phycocyanin polypeptide, is digested using ultrasonic wave added, and ultrasonic wave has cavitation, generation
Bubble ruptures through extruding, can produce extremely strong mechanical shear stress with moment, makes protein degradation or changes protein conformation, promotees
Its hydrophilic radical is more exposed, improve solubility of the phycocyanin in water, be conducive to enzyme and phycocyanin substrate
With reference to, can within a short period of time significantly improve the enzymolysis efficiency of phycocyanin, resulting polypeptide has stronger anti-oxidant
Property.
It is below specific embodiment part.
Embodiment 1
(1) 5g phycocyanins (A is weighed620/A280>2) it is put into container, 200mL distilled water is added, in temperature constant magnetic stirring
30min is stirred on device, it is fully dissolved, obtain phycocyanin solution；
(2) the phycocyanin solution obtained by step (1) is adjusted into pH to 6.0, is subsequently adding 1.5g papain (enzyme activity
Power 8 × 105U/g), ultrasonically treated in water bath sonicator device after stirring, ultrasonic power is 600W, 55 DEG C of reaction temperature,
Reaction time is 50min, obtains enzymolysis product；
(3) enzyme digestion reaction product is brought rapidly up to 90 DEG C of insulation 10min, 30 DEG C, 10000r/min is cooled to after the enzyme that goes out
Centrifugation 10min, takes supernatant, as enzymolysis liquid, and the degree of hydrolysis for measuring phycocyanin is 13.68%；
(4) use trapped molecular weight carries out hyperfiltration treatment for 5kD milipore filters by step (3) gained enzymolysis liquid, is surpassed
Filtrate；
(5) step (4) gained ultrafiltrate is concentrated in vacuo, spray drying obtains phycocyanin polypeptide.Various concentrations
Phycocyanin polypeptide to DPPH radical scavenging activities as shown in figure 1, a curve in figure is phycocyanin peptide concentration
With the relation curve of the clearance rate of DPPH free radicals, another curve is the relation of the clearance rate of the concentration and DPPH free radicals of VC
Curve.It can be seen that, phycocyanin polypeptide has certain relation to the clearance rate and concentration of DPPH free radicals, when phycocyanin polypeptide
When concentration reaches 5mg/mL, DPPH free radical scavenging activities are up to 87.31%.
Embodiment 2
(1) 5g phycocyanins (A is weighed620/A280>2) it is put into container, 50mL distilled water is added, in temperature constant magnetic stirring
Stirred on device to fully dissolving, obtain phycocyanin solution；
(2) the phycocyanin solution obtained by step (1) is adjusted into pH to 5.0, then by 5% addition of phycocyanin weight
Papain (enzyme activity 10 × 105U/g), ultrasonically treated in water bath sonicator device after stirring, ultrasonic power is
300W, 50 DEG C of reaction temperature, the reaction time is 30min, obtains enzymolysis product；
(3) enzyme digestion reaction product is brought rapidly up to 85 DEG C of insulation 15min, 30 DEG C, 10000r/min is cooled to after the enzyme that goes out
Centrifugation 10min, takes supernatant, as enzymolysis liquid, and the degree of hydrolysis for measuring phycocyanin is 12.97%；
(4) use trapped molecular weight carries out hyperfiltration treatment for 10kD milipore filters by step (3) gained enzymolysis liquid, is surpassed
Filtrate；
(5) step (4) gained ultrafiltrate is concentrated in vacuo, spray drying obtains phycocyanin polypeptide.Gained algae is blue
Polypeptide has certain relation to the clearance rate and concentration of DPPH free radicals, when the concentration of phycocyanin polypeptide reaches 5mg/mL
When, DPPH free radical scavenging activities are up to 80.98%.
Embodiment 3
(1) 10g phycocyanins (A is weighed620/A280>2) it is put into container, adds 500mL distilled water, is stirred in constant temperature magnetic force
Mix and stirred on device to fully dissolving, obtain phycocyanin solution；
(2) the phycocyanin solution obtained by step (1) is adjusted into pH to 7.0, then by 1% addition of phycocyanin weight
Papain (enzyme activity 10 × 105U/g), ultrasonically treated in water bath sonicator device after stirring, ultrasonic power is
800W, 60 DEG C of reaction temperature, the reaction time is 50min, obtains enzymolysis product；
(3) enzyme digestion reaction product is brought rapidly up to 95 DEG C of insulation 5min, is cooled to 30 DEG C after the enzyme that goes out, 10000r/min from
Heart 10min, takes supernatant, as enzymolysis liquid, and the degree of hydrolysis for measuring phycocyanin is 13.59%；
(4) use trapped molecular weight carries out hyperfiltration treatment for 1kD milipore filters by step (3) gained enzymolysis liquid, is surpassed
Filtrate；
(5) step (4) gained ultrafiltrate is concentrated in vacuo, spray drying obtains phycocyanin polypeptide.Gained algae is blue
Polypeptide has certain relation to the clearance rate and concentration of DPPH free radicals, when the concentration of phycocyanin polypeptide reaches 5mg/mL
When, DPPH free radical scavenging activities are up to 85.16%.
Embodiment 4
(1) 5g phycocyanins (A is weighed620/A280>2) it is put into container, 200mL distilled water is added, in temperature constant magnetic stirring
30min is stirred on device, it is fully dissolved, obtain phycocyanin solution；
(2) the phycocyanin solution obtained by step (1) is adjusted into pH to 6.0, then by 3% addition of phycocyanin weight
Neutral proteinase (enzyme activity 3 × 105U/g), ultrasonically treated in water bath sonicator device after stirring, ultrasonic power is
600W, 55 DEG C of reaction temperature, the reaction time is 50min, obtains enzymolysis product；
(3) enzyme digestion reaction product is brought rapidly up to 95 DEG C of insulation 5min, is cooled to 30 DEG C after the enzyme that goes out, 10000r/min from
Heart 10min, takes supernatant, as enzymolysis liquid, and the degree of hydrolysis for measuring phycocyanin is 11.82%；
(4) use trapped molecular weight carries out hyperfiltration treatment for 1kD milipore filters by step (3) gained enzymolysis liquid, is surpassed
Filtrate；
(5) step (4) gained ultrafiltrate is concentrated in vacuo, spray drying obtains phycocyanin polypeptide.Gained algae is blue
Polypeptide has certain relation to the clearance rate and concentration of DPPH free radicals, when the concentration of phycocyanin polypeptide reaches 6mg/mL
When, DPPH free radical scavenging activities are up to 75.82%.
Embodiment 5
(1) 5g phycocyanins (A is weighed620/A280>2) it is put into container, 50mL distilled water is added, in temperature constant magnetic stirring
Stirred on device to fully dissolving, obtain phycocyanin solution；
(2) the phycocyanin solution obtained by step (1) is adjusted into pH to 7.0, then by 5% addition of phycocyanin weight
Neutral proteinase (enzyme activity 1 × 105U/g), ultrasonically treated in water bath sonicator device after stirring, ultrasonic power is
800W, 45 DEG C of reaction temperature, the reaction time is 30min, obtains enzymolysis product；
(3) enzyme digestion reaction product is brought rapidly up to 85 DEG C of insulation 15min, 40 DEG C, 10000r/min is cooled to after the enzyme that goes out
Centrifugation 10min, takes supernatant, as enzymolysis liquid, and the degree of hydrolysis for measuring phycocyanin is 11.06%；
(4) use trapped molecular weight carries out hyperfiltration treatment for 10kD milipore filters by step (3) gained enzymolysis liquid, is surpassed
Filtrate；
(5) step (4) gained ultrafiltrate is concentrated in vacuo, spray drying obtains phycocyanin polypeptide.Gained algae is blue
Polypeptide has certain relation to the clearance rate and concentration of DPPH free radicals, when the concentration of phycocyanin polypeptide reaches 6mg/mL
When, DPPH free radical scavenging activities are up to 70.03%.
The above is only the preferred embodiment of invention, it is noted that for those skilled in the art,
On the premise of inventive principle is not departed from, some improvements and modifications can also be made, these improvements and modifications also should be regarded as invention
Protection domain.
Claims (10)
1. a kind of preparation method of phycocyanin polypeptide, it is characterised in that comprise the following steps：
Add water in phycocyanin, stirring makes it fully dissolve, and obtains phycocyanin solution；
To addition papain or neutral proteinase in the phycocyanin solution, after stirring, ultrasonically treated 30~
50min, obtains enzymolysis product；
The enzymolysis product is warming up to 85 DEG C~95 DEG C and 5~15min is incubated, cooled down after the enzyme that goes out, centrifugation takes supernatant, as
Enzymolysis liquid；
The enzymolysis liquid is carried out into hyperfiltration treatment, ultrafiltrate is obtained；
The ultrafiltrate is concentrated in vacuo, spray drying treatment obtains the phycocyanin polypeptide.
2. the preparation method of phycocyanin polypeptide as claimed in claim 1, it is characterised in that in adding water to phycocyanin,
Stirring makes it fully dissolve, and obtains in the operation of phycocyanin solution, and the solid-liquid ratio of phycocyanin and water is 1:10~1:50.
3. the preparation method of phycocyanin polypeptide as claimed in claim 1, it is characterised in that in adding water to phycocyanin,
Stirring makes it fully dissolve, and obtains in the operation of phycocyanin solution, and using temperature constant magnetic stirring, temperature is 50 DEG C~55 DEG C.
4. the preparation method of phycocyanin polypeptide as claimed in claim 1, it is characterised in that papain and phycocyanin
Mass ratio be 1:100~1:20.
5. the preparation method of phycocyanin polypeptide as claimed in claim 1, it is characterised in that neutral proteinase and phycocyanin
Mass ratio be 1:30~1:20.
6. the preparation method of phycocyanin polypeptide as claimed in claim 1, it is characterised in that add papain it
Before, also including the pH of the phycocyanin solution is adjusted to 5.0~7.0 the step of.
7. the preparation method of phycocyanin polypeptide as claimed in claim 1, it is characterised in that add neutral proteinase it
Before, also including the pH of the phycocyanin solution is adjusted to 6.0~7.0 the step of.
8. the preparation method of phycocyanin polypeptide as claimed in claim 1, it is characterised in that add papain to be surpassed
During sonication, reaction temperature is 50 DEG C~60 DEG C.
9. the preparation method of phycocyanin polypeptide as claimed in claim 1, it is characterised in that add neutral proteinase to be surpassed
During sonication, reaction temperature is 45 DEG C~55 DEG C.
10. the preparation method of phycocyanin polypeptide as claimed in claim 1, it is characterised in that in ultrasonically treated operation, surpasses
Acoustical power is 300~800W.
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| CN108077941A (en) * | 2017-12-22 | 2018-05-29 | 广东正当年生物科技有限公司 | A kind of algae peptide combinations and preparation method and application |
| CN109096548A (en) * | 2018-07-11 | 2018-12-28 | 天津商业大学 | A method of utilizing phycocyanin and its hydrolysate preparation retrogradation resistant starch |
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| CN110106218A (en) * | 2019-04-24 | 2019-08-09 | 北海生巴达生物科技有限公司 | A kind of algae small-molecular peptides extracting method |
| CN110241164A (en) * | 2019-06-26 | 2019-09-17 | 厦门昶科生物工程有限公司 | A kind of extracting method of blood red Euglena polypeptide |
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| CN113293190A (en) * | 2021-07-27 | 2021-08-24 | 中国科学院烟台海岸带研究所 | Phycobilin binding peptides and uses thereof |
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| CN115716870A (en) * | 2022-11-21 | 2023-02-28 | 北京工商大学 | Phycocyanin anti-tumor peptide and preparation method and application thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108077941A (en) * | 2017-12-22 | 2018-05-29 | 广东正当年生物科技有限公司 | A kind of algae peptide combinations and preparation method and application |
| CN109096548B (en) * | 2018-07-11 | 2021-06-01 | 天津商业大学 | Method for preparing retrogradation resistant starch by using phycocyanin and hydrolysate thereof |
| CN109096548A (en) * | 2018-07-11 | 2018-12-28 | 天津商业大学 | A method of utilizing phycocyanin and its hydrolysate preparation retrogradation resistant starch |
| CN109680028A (en) * | 2019-01-08 | 2019-04-26 | 广东海洋大学 | A kind of deliquescent method of improvement aquatic product protein |
| CN110106218A (en) * | 2019-04-24 | 2019-08-09 | 北海生巴达生物科技有限公司 | A kind of algae small-molecular peptides extracting method |
| CN110241164A (en) * | 2019-06-26 | 2019-09-17 | 厦门昶科生物工程有限公司 | A kind of extracting method of blood red Euglena polypeptide |
| CN112226476A (en) * | 2020-10-10 | 2021-01-15 | 暨南大学 | Enzymolysis method of phycocyanin and application thereof |
| CN113355383A (en) * | 2021-06-04 | 2021-09-07 | 中国水产科学研究院黄海水产研究所 | Comprehensive utilization and processing method of spirulina |
| CN113293190A (en) * | 2021-07-27 | 2021-08-24 | 中国科学院烟台海岸带研究所 | Phycobilin binding peptides and uses thereof |
| CN113896765A (en) * | 2021-09-27 | 2022-01-07 | 青岛科技大学 | Antioxidant peptide and preparation method and application thereof |
| CN113896765B (en) * | 2021-09-27 | 2023-06-20 | 青岛科技大学 | Antioxidant peptide and preparation method and application thereof |
| CN115716870A (en) * | 2022-11-21 | 2023-02-28 | 北京工商大学 | Phycocyanin anti-tumor peptide and preparation method and application thereof |
| CN115716870B (en) * | 2022-11-21 | 2023-10-20 | 北京工商大学 | Phycocyanin anti-tumor peptide and preparation method and application thereof |
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| CN106701874B (en) | 2023-05-16 |
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