CN104694603A - Method for preparing laver polypeptide stock solution - Google Patents
Method for preparing laver polypeptide stock solution Download PDFInfo
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- CN104694603A CN104694603A CN201510077307.6A CN201510077307A CN104694603A CN 104694603 A CN104694603 A CN 104694603A CN 201510077307 A CN201510077307 A CN 201510077307A CN 104694603 A CN104694603 A CN 104694603A
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Abstract
The invention discloses a method for preparing a laver polypeptide stock solution. The method comprises the following steps: taking porphyra haitanensis as a raw material, drying, grinding, adding water for soaking, grinding, and performing other treatments, thereby obtaining laver particle slurry with fine particles; adding a certain amount of protease for performing secondary enzymolysis, thereby obtaining enzymatic hydrolysate; and performing enzyme deactivation and centrifugal treatment on the enzymatic hydrolysate, thereby obtaining the laver polypeptide stock solution. With the adoption of the secondary enzymolysis, the laver is subjected to full enzymolysis, the enzymolysis time is shortened, and the extraction rate of the laver polypeptide stock solution is greatly improved.
Description
Technical field
The present invention relates to the technical field of bioengineering field, particularly relate to a kind of preparation method of laver polypeptide stoste.
Background technology
Laver contain up to about 30% protein and iodine, multivitamin and inorganic salts, delicious flavour can, in order to treat thyromegaly and to reduce cholesterol, be also a kind of important economical alga except edible, be distributed widely in all over the world, but based on temperate zone.
The processing mode of current laver is more single, mainly makes laver dry products, needs the process such as bubble is sent out, the cooking, or form sea sedge through seasoning process after being baked by laver time edible.Therefore, some investigators have probed into the mode of laver deep processing further, by extracting the nutritive ingredient in laver, in order to prepare natural product, or develop beverage based on this.Research proves, laver contains abundant laver polypeptide, and its tool has efficacy in lowering high blood pressure, and for this reason, extracts and prepares the focus that laver polypeptide also becomes research.
In the prior art, usually adopt proteasome degradation method to prepare laver polypeptide, the enzymolysis time of proteolytic enzyme is more than 5 hours, and the throw out after degraded is simply discarded.The present inventor is through a large amount of research and development and practice, once carrying out in proteasome degradation experiment to laver, throw out is analyzed, find also there is the laver substrate be not easily degraded by proteases in a large number, the present inventor attempts increase albumen enzyme amount and extend enzymolysis time again, but final throw out still also has the laver substrate be not degraded in a large number, that is, in a proteolysis process, no matter be increase albumen enzyme amount or extend enzymolysis time, for the extraction yield of laver polypeptide, the amplitude of increase is all very limited.Therefore the explanation that have also been obtained " reasonable " is abandoned in a large number after throw out enzymolysis.In view of this, the present invention studies and devises a kind of preparation method of laver polypeptide stoste, and this case produces thus.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of laver polypeptide stoste, by proteolytic enzyme secondary edman degradation Edman, to improve the extraction yield of laver polypeptide further.
For achieving the above object, the present invention solves the technical scheme of its technical problem and is:
A preparation method for laver polypeptide stoste, comprises the following steps:
Step one, prepare the homogenate of laver particle:
Be raw material with porphyra haitanensis, pulverize, form dry laver powder after oven dry with high speed disintegrator, the add water immersion of spending the night of described dry laver powder makes it fully swelling, then grinds further with sand mill, becomes the laver particle homogenate that particle is tiny;
Step 2, employing secondary enzyme process enzymolysis:
During first time enzymolysis, using the homogenate of described laver particle as substrate, by proteolytic enzyme by mass percentage 1.5%, with described dry laver powder weighing scale, add in described substrate, adjust pH to 2-9, enzymolysis 3h at 35 ~ 60 DEG C, obtain enzymolysis solution L1, by described enzymolysis solution L1 centrifugal 30min under 15000 × g, 4 DEG C of conditions, obtain supernatant liquor S1 and throw out S2;
During second time enzymolysis, by mass volume ratio 1:10, with described dry laver powder weighing scale, throw out S2 is added to the water and makes it resuspended, obtain laver suspension, by proteolytic enzyme by mass percentage 1.0%, with described dry laver powder weighing scale, add in described laver suspension, adjust pH to 2-9, enzymolysis 2h at 35 ~ 60 DEG C, obtain enzymolysis solution L2, by described enzymolysis solution L2 at 8000 × g, centrifugal 15min under 4 DEG C of conditions, obtain supernatant liquor S3 and throw out S4, discard described throw out S4, collect, merge described supernatant liquor S1 and described supernatant liquor S3, obtain supernatant liquor S5,
Step 3, separation and purification:
Described supernatant liquor S5 is boiled in boiling water bath 10min to go out enzyme, centrifugal 15min under 8000 × g, 4 DEG C of conditions, obtains supernatant liquor S6 and throw out S7, discards described throw out S7, collect described supernatant liquor S6, be laver polypeptide stoste afterwards.
As the optimal way of embodiment, in described step one, described dry laver powder is added to the water according to mass volume ratio 1:15, immersion of spending the night at 4 DEG C.
As the optimal way of embodiment, in described step one, the rotating speed of described sand mill is 2000r/min, and milling time is 80min.
As the optimal way of embodiment, described proteolytic enzyme is the combination of papoid, bromeline, trypsinase, stomach en-, neutral protease, Sumizyme MP or food flavor enzyme and above-mentioned enzyme.
The invention has the beneficial effects as follows: adopt multiple fragmentation, enzymolysis process, carry out shattering, grinding after laver is dried, make laver cell fully broken, be conducive to content fully to discharge, utilize prozyme to carry out secondary enzymolysis, further protein transduction residual in centrifugal rear laver slag is changed into polypeptide, improve polypeptide extraction yield, take full advantage of laver raw material, cost-saving, and simple to operate.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.
The present invention take porphyra haitanensis as raw material, through drying, pulverize, soak, the process such as grinding, obtain the laver particle homogenate that particle is tiny, add a certain amount of proteolytic enzyme secondary enzymolysis, obtain enzymolysis solution, namely enzymolysis solution obtains laver polypeptide stoste after the enzyme that goes out, centrifugal treating.
By protein content in the first method Kjeldahl nitrogen determination laver stoste in GB 50095-2010, to assess the extraction yield of laver polypeptide.The hypotensive activity of laver polypeptide is reacted by the body outer suppressioning experiment of angiotensin transferase (ACE).
The mensuration of laver polypeptide hypotensive activity
The 0.1U/ml ACE of the 5mM reaction substrate Hip-His-Leu of (1) 10 μ l, 10 μ l and the 0.2M borate buffer (pH8.3, containing 0.3 M NaCl) of 10 μ l constitute external ACE catalystic converter system.React 30min at 37 DEG C after, add the 1M hydrochloric acid termination reaction of 25 μ l.In reaction system, add the ethyl acetate of 150 μ l again, vibrate and within 5 minutes, make it fully mix, under 1000 r/min after centrifugal 5min, taking out 100 μ l ester layer moves in another PCR pipe, dry up with blower, the deionized water adding 100 μ l dissolves again, under 228nm, measure light absorption value.Replace the damping fluid of 10 μ l by the laver polypeptide stoste of 10 μ l, step is the same, measures light absorption value.
(2) ACE inhibiting rate (%)=(A-B+D)/(A-C) × 100%.Wherein, A is the light absorption value of unrestraint agent pipe, and B is the light absorption value containing inhibitor pipe, and C is the light absorption value of blank pipe, and D is the light absorption value of inhibitor blank tube.
the pre-treatment of embodiment 1 laver
Take 1kg porphyra haitanensis and dry 2h at 50 DEG C, the dry laver obtained is put into high speed disintegrator and pulverizes, then according to mass volume ratio 1:15(with dry laver powder weighing scale) dry laver powder is added to the water, immersion of spending the night at 4 DEG C.Under rotating speed is 2000r/min condition, grind 80min with sand mill again, obtain the homogenate of laver particle.
embodiment 2 secondary enzymolysis
Take the homogenate of 64g laver particle and (include the dry laver powder of 4g and 60g water, i.e. 1:15 grinding), add 60mg trypsinase, mix, adjust pH to 8 with the sodium carbonate solution of 1mol/l and 0.1mol/l, enzymolysis 3h at 37 DEG C, obtains enzymolysis solution L1, by enzymolysis solution L1 centrifugal 30min under 15000 × g, 4 DEG C of conditions, obtain supernatant liquor S1 and throw out S2.Throw out S2 is added in 40ml water and makes it resuspended, 40mg Sumizyme MP (Sumizyme MP is obtained by Bacillus licheniformis normal fermentation) is added in re-suspension liquid, mix, pH to 8 is adjusted with the sodium carbonate solution of 1mol/l and 0.1mol/l, enzymolysis 2h at 50 DEG C, obtain enzymolysis solution L2, by enzymolysis solution L2 centrifugal 15min under 8000 × g, 4 DEG C of conditions, obtain supernatant liquor S3 and throw out S4.Discard throw out S4, collect, merge supernatant liquor S1 and S3, obtain supernatant liquor S5.
embodiment 3 separation and purification
S5 is boiled in boiling water bath 10min to go out enzyme, centrifugal 15min under 8000 × g, 4 DEG C of conditions, obtains supernatant liquor S6 and throw out S7, discards throw out S7 afterwards, collects supernatant liquor S6, is laver polypeptide stoste.
After measured, in laver polypeptide stoste, protein content is 1.16%, and extraction yield is 71.6%, ACE maximum inhibition is 93.3%.
The present invention's first time enzymolysis time is 3 hours, second time enzymolysis time is 2 hours, first time, enzymolysis was after 3 hours, the present inventor finds the hydrolysis of the laver polypeptide meeting protease inhibition generated, namely the output of laver polypeptide reaches the stage of stable development, even if extend enzymolysis time again, the amount of laver polypeptide also can not increase again, therefore, the throw out of the present inventor to first time enzymolysis has carried out second time enzymolysis, because this throw out is not substantially containing laver polypeptide, thus in second time enzymolysis, relieve the effect of Product inhibiton, the laver substrate contained in throw out is made to be decomposed utilization further.
To sum up, the present inventor adopts secondary enzymolysis method, overcomes Product inhibiton effect, while hinge structure Reaction time shorten, substantially increases the extraction yield of laver polypeptide.All distortion that those of ordinary skill in the art can directly derive from the disclosure of invention or associate, all should think protection scope of the present invention.
Claims (4)
1. a preparation method for laver polypeptide stoste, is characterized in that: comprise the following steps:
Step one, prepare the homogenate of laver particle:
Be raw material with porphyra haitanensis, pulverize, form dry laver powder after oven dry with high speed disintegrator, the add water immersion of spending the night of described dry laver powder makes it fully swelling, then grinds further with sand mill, becomes the laver particle homogenate that particle is tiny;
Step 2, employing secondary enzyme process enzymolysis:
During first time enzymolysis, using the homogenate of described laver particle as substrate, by proteolytic enzyme by mass percentage 1.5%, with described dry laver powder weighing scale, add in described substrate, adjust pH to 2-9, enzymolysis 3h at 35 ~ 60 DEG C, obtain enzymolysis solution L1, by described enzymolysis solution L1 centrifugal 30min under 15000 × g, 4 DEG C of conditions, obtain supernatant liquor S1 and throw out S2;
During second time enzymolysis, by mass volume ratio 1:10, with described dry laver powder weighing scale, throw out S2 is added to the water and makes it resuspended, obtain laver suspension, by proteolytic enzyme by mass percentage 1.0%, with described dry laver powder weighing scale, add in described laver suspension, adjust pH to 2-9, enzymolysis 2h at 35 ~ 60 DEG C, obtain enzymolysis solution L2, by described enzymolysis solution L2 at 8000 × g, centrifugal 15min under 4 DEG C of conditions, obtain supernatant liquor S3 and throw out S4, discard described throw out S4, collect, merge described supernatant liquor S1 and described supernatant liquor S3, obtain supernatant liquor S5,
Step 3, separation and purification:
Described supernatant liquor S5 is boiled in boiling water bath 10min to go out enzyme, centrifugal 15min under 8000 × g, 4 DEG C of conditions, obtains supernatant liquor S6 and throw out S7, discards described throw out S7, collect described supernatant liquor S6, be laver polypeptide stoste afterwards.
2. the preparation method of a kind of laver polypeptide stoste as claimed in claim 1, is characterized in that: in described step one, is added to the water by described dry laver powder according to mass volume ratio 1:15, immersion of spending the night at 4 DEG C.
3. the preparation method of a kind of laver polypeptide stoste as claimed in claim 1, is characterized in that: in described step one, and the rotating speed of described sand mill is 2000r/min, and milling time is 80min.
4. the preparation method of a kind of laver polypeptide stoste as claimed in claim 1, is characterized in that: described proteolytic enzyme is the combination of papoid, bromeline, trypsinase, stomach en-, neutral protease, Sumizyme MP or food flavor enzyme and above-mentioned enzyme.
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| CN201510077307.6A CN104694603A (en) | 2015-02-13 | 2015-02-13 | Method for preparing laver polypeptide stock solution |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070022A (en) * | 2018-02-09 | 2018-05-25 | 海欣食品股份有限公司 | A kind of seaweed antifreeze peptide concentrate and preparation method thereof |
| CN109852656A (en) * | 2019-03-21 | 2019-06-07 | 大洲新燕(厦门)生物科技有限公司 | A kind of bird's nest polypeptide powder and preparation method thereof |
| CN110613128A (en) * | 2019-07-24 | 2019-12-27 | 宁波华子制药设备制造有限公司 | Low-temperature extraction production process of active sheep placenta |
| CN113575750A (en) * | 2021-07-08 | 2021-11-02 | 大连工业大学 | Method for extracting laver protein and its product |
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| CN1687444A (en) * | 2005-03-23 | 2005-10-26 | 江苏大学 | Method for preparing peptide of decrease blood pressure in laver by using enzyme-membrane coupling technique and application thereof |
| CN102154423A (en) * | 2011-01-12 | 2011-08-17 | 淮海工学院 | Method for preparing active peptide of laver |
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2015
- 2015-02-13 CN CN201510077307.6A patent/CN104694603A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1687444A (en) * | 2005-03-23 | 2005-10-26 | 江苏大学 | Method for preparing peptide of decrease blood pressure in laver by using enzyme-membrane coupling technique and application thereof |
| CN102154423A (en) * | 2011-01-12 | 2011-08-17 | 淮海工学院 | Method for preparing active peptide of laver |
Non-Patent Citations (2)
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| 刘淑集 等: "坛紫菜降血压活性肽的分离纯化及分子质量测定", 《食品科学》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070022A (en) * | 2018-02-09 | 2018-05-25 | 海欣食品股份有限公司 | A kind of seaweed antifreeze peptide concentrate and preparation method thereof |
| CN108070022B (en) * | 2018-02-09 | 2020-12-25 | 海欣食品股份有限公司 | Seaweed antifreeze polypeptide concentrated solution and preparation method thereof |
| CN109852656A (en) * | 2019-03-21 | 2019-06-07 | 大洲新燕(厦门)生物科技有限公司 | A kind of bird's nest polypeptide powder and preparation method thereof |
| CN110613128A (en) * | 2019-07-24 | 2019-12-27 | 宁波华子制药设备制造有限公司 | Low-temperature extraction production process of active sheep placenta |
| CN113575750A (en) * | 2021-07-08 | 2021-11-02 | 大连工业大学 | Method for extracting laver protein and its product |
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Effective date of registration: 20160323 Address after: 041000 No. 130 Jiefang East Road, Shanxi, Linfen Applicant after: Shanxi Connaught Biotechnology Co., Ltd. Address before: Yinjiang road in Jimei District of Xiamen City, Fujian Province, No. 185 361000 Applicant before: Jimei University |
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Application publication date: 20150610 |