CN103060408A - Method for preparing purple perilla oligopeptide with high F value through immobilization enzymatic preparation - Google Patents
Method for preparing purple perilla oligopeptide with high F value through immobilization enzymatic preparation Download PDFInfo
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- CN103060408A CN103060408A CN2012105577779A CN201210557777A CN103060408A CN 103060408 A CN103060408 A CN 103060408A CN 2012105577779 A CN2012105577779 A CN 2012105577779A CN 201210557777 A CN201210557777 A CN 201210557777A CN 103060408 A CN103060408 A CN 103060408A
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Abstract
The invention provides a novel craft for preparing purple perilla oligopeptide with high F value through immobilization enzymatic preparation, and an immobilization enzyme method is adopted to prepare the purple perilla oligopeptide with the high F value. Firstly, fructus perilla which is unshelled and smashed is adopted to be blended with water to flow into a constant stirring tank reactor filled with a certain amount of immobilization incision enzyme, after the reaction is carried out for some time, and enzymatic hydrolysate is guided to flow out to be carried out with enzyme deactivation centrifugation to extract perilla oil, polypeptide liquid and residues. Secondly, the polypeptide liquid flows into the other constant stirring tank reactor filled with a certain amount of immobilization excision enzyme, after the reaction is carried out for some time, the enzymatic hydrolysate is guided to flow out to be carried out with the enzyme deactivation centrifugation to extract oligopeptide liquid, activated carbon adsorption is added to the oligopeptide liquid to remove aromatic amino acid, and separation and purification are further carried out to produce the oligopeptide with the high F value.
Description
Technical field
What the present invention relates to is a kind of production method of functional foodstuff, and specifically a kind of immobilized enzyme method prepares the method for high F value purple perilla oligopeptide.
Background technology
Purple perilla is a kind of traditional drugs food dual purpose plant, its fruit and perilla seed are the high oil resources of a kind of oil length simultaneously, be usually used at present extracting perilla oil, its by product purple perilla dregs of rice are usually used in doing the animal feed, and the research of producing synchronously perilla oil and purple perilla albumen or polypeptide products is not yet arranged.
The present invention is take perilla seed as raw material, decortication is pulverized rear and distilled water is mixed in proportion, use first the restriction endonuclease of energy protein hydrolysate specific site, downcut the N-end of peptide section or the die aromatischen Aminosaeuren of C-end, enzymolysis is by the centrifugal perilla oil that obtains of the enzyme that goes out, the polypeptide liquid and the residue that mix, then add another in the polypeptide liquid and can downcut the excision enzyme of die aromatischen Aminosaeuren group, the die aromatischen Aminosaeuren residue is discharged from peptide chain, centrifugal through the enzyme that goes out, the charcoal absorption purifying obtains the oligopeptide mixed solution, again through dialysis, ultrafiltration, lyophilize obtains purity up to 90% high F value oligopeptide.The immobilized enzyme that restriction endonuclease, the excision enzyme that uses all uses oily Crosslinking method to make has increased recovery rate and the repeating utilization factor of enzyme, has reduced the wasting of resources.
Summary of the invention
The invention provides a kind of novel high F value purple perilla oligopeptide technology of preparing, the perilla seed that namely at first adopts shelling to pulverize, mix the rear continuous agitator tank reactor that a certain amount of immobilization restriction endonuclease is housed that flows into water, after reaction for some time that enzymolysis solution outflow and the enzyme that goes out is centrifugal, isolate perilla oil, polypeptide liquid, residue; Polypeptide liquid is flowed into the continuous agitator tank reactor that another is equipped with a certain amount of immobilization excision enzyme, after reaction for some time that enzymolysis solution outflow and the enzyme that goes out is centrifugal, separate and obtain oligopeptide solution, add charcoal absorption and remove die aromatischen Aminosaeuren, can obtain high F value oligopeptide through further separation and purification.
A kind of immobilized enzyme method prepares the method for high F value purple perilla oligopeptide, and method steps is as follows:
Step 1: shelling is pulverized to perilla seed, is mixed to get mixed solution with water, and described amount of water is 6 times of quality of material.
Step 2: gained mixed solution in the step 1 is put into the continuous agitator tank reactor that immobilization Alcalase2.4L Sumizyme MP is housed, flow out product behind the reaction 3h and carry out centrifugally, isolate perilla oil, polypeptide liquid and residue.
Step 3: the polypeptide liquid that step 2 is collected is put into immobilized papain continuous agitator tank reactor is housed, and flows out the product enzyme that goes out behind the reaction 2h, and high speed centrifugation is isolated mixed solution.
Step 4: the mixed solution in the step 3 is added a certain amount of gac, and die aromatischen Aminosaeuren is removed in absorption, obtains the purple perilla oligopeptide of high F value through the ultrafiltration desalination.
The grinding particle size of perilla seed is at the 60-90 order in the described step 1.
In the described step 2 immobilization Alcalase2.4L Sumizyme MP by 3% chitosan, 0.5% glutaraldehyde behind Crosslinking method crosslinking time 4h, transfer pH=9 immobilization 8h on to the carrier of enzyme amount 950u/g to make, its addition is the 0.5-3% of concentration of substrate, temperature 40-60 ℃, pH=7.5-9.5, the enzyme condition of going out is to heat 5min in the boiling water for the enzyme condition of going out, centrifugal condition is rotating speed 4500rpm, 4 ℃ of temperature, time 20min.
The same Alcalase2.4L of the preparation method of immobilized papain in the described step 3, addition is the 0.5-3% of concentration of substrate, temperature 40-60 ℃, pH=7.5-9.5, the enzyme condition of going out is to heat 5min in the boiling water for the enzyme condition of going out, centrifugal condition is rotating speed 4500rpm, 4 ℃ of temperature, time 20min.
In the described step 4 gac addition be the substrate quality 5-15 doubly, pH=2-5, temperature is 25-50 ℃, the ultra-filtration membrane aperture is the 2-8 micron, molecular weight cut-off is at 100-1000, operation pressure reduction is at 1-2.5MPa.
Useful point of the present invention is: the present invention adopts a kind of immobilized enzyme method to prepare high F value purple perilla oligopeptide, the perilla seed that at first adopts shelling to pulverize, mix the rear continuous agitator tank reactor that a certain amount of immobilization restriction endonuclease is housed that flows into water, after reaction for some time that enzymolysis solution outflow and the enzyme that goes out is centrifugal, isolate perilla oil, polypeptide liquid, residue; Polypeptide liquid is flowed into the continuous agitator tank reactor that another is equipped with a certain amount of immobilization excision enzyme, after reaction for some time that enzymolysis solution outflow and the enzyme that goes out is centrifugal, separate and obtain oligopeptide solution, add charcoal absorption and remove die aromatischen Aminosaeuren, can obtain high F value oligopeptide through further separation and purification.
Description of drawings
Accompanying drawing is process route chart of the present invention
Embodiment:
Embodiment one: the step of present embodiment is as follows:
Step 1: shelling is pulverized to perilla seed, and grinding particle size is 60 orders, mixes in the ratio of 1:6 with water.
Step 2: gained material in the step 1 is put into the continuous agitator tank reactor that immobilization Alcalase2.4L Sumizyme MP is housed, and it is centrifugal to flow out the product enzyme that goes out behind the reaction 3h, isolates perilla oil, polypeptide liquid and residue.The addition of immobilization Alcalase2.4L Sumizyme MP is 1% of concentration of substrate, 55 ℃ of temperature, pH=8.5; The enzyme condition of going out is to heat 5min in the boiling water; Centrifugal condition is rotating speed 4500rpm, 4 ℃ of temperature, time 20min.
Step 3: the polypeptide liquid that step 2 is collected is put into immobilized papain continuous agitator tank reactor is housed, and it is centrifugal to flow out the product enzyme that goes out behind the reaction 2h, isolates the oligopeptide mixed solution.The addition of immobilized papain is 1% of concentration of substrate, temperature 50 C, pH=6; The enzyme condition of going out is to heat 5min in the boiling water; Centrifugal condition is rotating speed 8000rpm, 4 ℃ of temperature, time 10min.
Step 4: with the ratio adding gac of the oligopeptide mixed solution in the step 3 in 1:5, absorption 40min, pH=3,20 ℃ of temperature are removed die aromatischen Aminosaeuren.
Step 5: the material in the step 4 is obtained peptide molecular weight at 100 ~ 1000 high F value oligopeptide through ultrafiltration.
Specific implementation method two: this implementation method is that with specific implementation method one difference the grinding particle size of perilla seed in the step 1 is at 70 orders.Other composition is identical with specific implementation method one with step;
Specific implementation method three: this implementation method is that the addition of Alcalase2.4L in the step 2 is 2%, temperature 50 C, pH=8 with specific implementation method one difference;
Specific implementation method four: this implementation method is that the addition of papoid in the step 3 is 2% of concentration of substrate, temperature 60 C, pH=6.5 with specific implementation method one difference.Other composition is identical with specific implementation method one with step;
Specific implementation method five: this implementation method is that with specific implementation method one difference the gac addition is 1:10 in the step 4, absorption 60min, pH=4,30 ℃ of temperature.Other composition is identical with specific implementation method one with step.
Claims (5)
1. an immobilized enzyme method prepares the method for high F value purple perilla oligopeptide, it is characterized in that described method steps is as follows:
Step 1: shelling is pulverized to perilla seed, is mixed to get mixed solution with water, and described amount of water is 6 times of quality of material;
Step 2: gained mixed solution in the step 1 is put into the continuous agitator tank reactor that immobilization Alcalase2.4L Sumizyme MP is housed, flow out product behind the reaction 3h and carry out centrifugally, isolate perilla oil, polypeptide liquid and residue;
Step 3: the polypeptide liquid that step 2 is collected is put into immobilized papain continuous agitator tank reactor is housed, and flows out the product enzyme that goes out behind the reaction 2h, and high speed centrifugation is isolated mixed solution;
Step 4: the mixed solution in the step 3 is added a certain amount of gac, and die aromatischen Aminosaeuren is removed in absorption, obtains the purple perilla oligopeptide of high F value through the ultrafiltration desalination.
2. a kind of immobilized enzyme method according to claim 1 prepares the method for high F value purple perilla oligopeptide, it is characterized in that the grinding particle size of perilla seed in the described step 1 is at the 60-90 order.
3. a kind of immobilized enzyme method according to claim 1 prepares the method for high F value purple perilla oligopeptide, it is characterized in that in the described step 2 immobilization Alcalase2.4L Sumizyme MP by 3% chitosan, 0.5% glutaraldehyde behind Crosslinking method crosslinking time 4h, transfer pH=9 immobilization 8h on to the carrier of enzyme amount 950u/g to make, its addition is the 0.5-3% of concentration of substrate, temperature 40-60 ℃, pH=7.5-9.5, the enzyme condition of going out is to heat 5min in the boiling water for the enzyme condition of going out, centrifugal condition is rotating speed 4500rpm, 4 ℃ of temperature, time 20min.
4. a kind of immobilized enzyme method according to claim 1 prepares the method for high F value purple perilla oligopeptide, the same Alcalase2.4L of preparation method that it is characterized in that immobilized papain in the described step 3, addition is the 0.5-3% of concentration of substrate, temperature 40-60 ℃, pH=7.5-9.5, the enzyme condition of going out is to heat 5min in the boiling water for the enzyme condition of going out, centrifugal condition is rotating speed 4500rpm, 4 ℃ of temperature, time 20min.
5. a kind of immobilized enzyme method according to claim 1 prepares the method for high F value purple perilla oligopeptide, it is characterized in that gac addition in the described step 4 be the substrate quality 5-15 doubly, pH=2-5, temperature is 25-50 ℃, the ultra-filtration membrane aperture is the 2-8 micron, molecular weight cut-off is at 100-1000, and operation pressure reduction is at 1-2.5MPa.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865971A (en) * | 2014-03-14 | 2014-06-18 | 中华全国供销合作总社杭州茶叶研究所 | Tea protein high Fischer value oligopeptide as well as preparation method and application thereof |
| CN106578403A (en) * | 2016-12-07 | 2017-04-26 | 江南大学 | Comprehensive utilization method for perilla seed meal |
| CN109757600A (en) * | 2019-02-13 | 2019-05-17 | 湖北瑞邦生物科技有限公司 | A kind of preparation method of purple perilla peptide |
-
2012
- 2012-12-20 CN CN2012105577779A patent/CN103060408A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 侯利霞等: "响应面法优化固定化碱性蛋白酶工艺", 《河南工业大学学报(自然科学版)》 * |
| 童波等: "紫苏高F值低聚肽的制备研究", 《食品科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865971A (en) * | 2014-03-14 | 2014-06-18 | 中华全国供销合作总社杭州茶叶研究所 | Tea protein high Fischer value oligopeptide as well as preparation method and application thereof |
| CN103865971B (en) * | 2014-03-14 | 2017-02-22 | 中华全国供销合作总社杭州茶叶研究所 | Tea protein high Fischer value oligopeptide as well as preparation method and application thereof |
| CN106578403A (en) * | 2016-12-07 | 2017-04-26 | 江南大学 | Comprehensive utilization method for perilla seed meal |
| CN106578403B (en) * | 2016-12-07 | 2020-12-01 | 江南大学 | Comprehensive utilization method of perilla seed meal |
| CN109757600A (en) * | 2019-02-13 | 2019-05-17 | 湖北瑞邦生物科技有限公司 | A kind of preparation method of purple perilla peptide |
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Application publication date: 20130424 |