CN110106218A - A kind of algae small-molecular peptides extracting method - Google Patents
A kind of algae small-molecular peptides extracting method Download PDFInfo
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- CN110106218A CN110106218A CN201910335310.1A CN201910335310A CN110106218A CN 110106218 A CN110106218 A CN 110106218A CN 201910335310 A CN201910335310 A CN 201910335310A CN 110106218 A CN110106218 A CN 110106218A
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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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- 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 present invention relates to a kind of polypeptide technical field, specifically a kind of algae small-molecular peptides extracting method.Algae is dissolved in ultrasonication after water by the present invention, overflow the beneficiating ingredients such as the albumen in algae, reusing laccase, alkali protease, Pullulanase, serine protein hydrolase, alternately enzymatic hydrolysis dissociates the albumen in algae sufficiently to broken mixed solution progress alkali, acid, it is dried after carrying out ultrafiltration again, method of the invention possesses high small-molecular peptides recovery rate, greatly improve utilization by algae rate, reduce material waste, possess faster extraction rate again, is a kind of efficient, simple, fast algae small-molecular peptides extracting method.
Description
Technical field
The present invention relates to a kind of polypeptide technical field, specifically a kind of algae small-molecular peptides extracting method.
Background technique
Small-molecular peptides are a kind of biochemical substances between amino acid and protein, it is smaller than protein molecular weight, and are compared
Amino acid molecular amount is big, is the segment of a protein.It is connected between more than two amino acid with peptide bond, " the amino of formation
Sour chain " or " amino acid string " are just called peptide.Wherein, 10-15 are referred to as polypeptide with the peptide that upper amino acid forms, and by 2 to 9
Amino acid composition is just called oligopeptides, is just called small-molecular peptides or small peptide by what 2 to 15 amino acid formed.Algae contains very
Various nutritional ingredients needed by human abundant, can be from a large amount of small-molecular peptides be wherein extracted, from the small-molecular peptides wherein extracted
The limitation that current algae extract dissolubility is poor, fishy smell is heavy, and more conducively human consumption can be broken through, there is preferably immune adjust
The functions such as section, anti-oxidant, blood pressure lowering, antibacterial.
In recent years, with the development of science and technology, continuous improvement of people's living standards, natural active matter and function
Peptide becomes the hot spot of current research, such as bacteriostatic peptide, anti-oxidation peptide, Antihypertensive Peptides are led in medicines and health protection, food processing and cosmetics
Domain has a wide range of applications space, however, the extractive technique route of general polypeptide be 1. first by the material containing protein into
Row pretreatment is as dissolved, ultrasonication, multigelation etc.；2. its supernatant is carried out enzymatic hydrolysis or double enzymolysis etc.；3. dialysis removes
Remove salinity；4. ultrafiltration；5. gel chromatography；6. high performance liquid chromatography and electrophoresis etc. just can get the peptide fragment needed.According to report
Road natural activity peptide molecular weight is mostly 5000D hereinafter, must carry out above-mentioned steps to obtain such micromolecule polypeptide
Research and analysis.These operating technologies require height, and instrument and equipment requires complexity, needs the time long, be exactly those skilled in the art
It is also required to the time of a few days or a few weeks, when the extraction that the technical staff not being familiar with will complete micromolecule polypeptide just needs longer
Between.More importantly the polypeptide of living cells passes through extracting and developing purification process very long in this way, partly lose original
Activity, cause to get half the result with twice the effort.
Therefore, finding efficient, the simple, fast algae small-molecular peptides extracting method of one kind is the task of top priority.
Summary of the invention
In order to solve the above technical problems existing in the prior art, the present invention provides a kind of efficient, simple, fast algae
Micromolecular peptide extracting method, particular content are as follows:
A kind of algae small-molecular peptides extracting method, includes the following steps:
(1) algae cleaned up is weighed, is dissolved with water, keeps algae evenly dispersed in water, obtains；
(2) aqueous solution for being mixed with algae is subjected to ultrasonication；
(3) the mixed solution ph after ultrasonication is adjusted to 9.2-9.8, laccase, alkali protease, Pu Lu is added thereto
Blue enzyme, stirs evenly, and digests 1-4h at 35-55 DEG C, by the enzyme-deactivating in mixed solution；
(4) the mixed liquor tune ph to 6.1-6.6 by enzyme deactivation after living, is added pepsin, digests 20- at 40-45 DEG C
40min, by the enzyme-deactivating in mixed solution；
(5) mixed liquor of the enzyme deactivation after living is subjected to ultrafiltration；
(6) by after ultrafiltration concentration, it is dry after to obtain the final product.
Preferably, in the step (1), solid-liquid ratio 20-60g/L is controlled.This solid-liquid ratio is relatively mild, is suitable for subsequent broken
Operation carries out.
Preferably, the ultrasonication of the step (2) is that it is broken to carry out ultrasound with the frequency of 20-40KHz at 0-10 DEG C
It is broken.The effect that this temperature and frequency carry out ultrasonication is preferable, is crushed fast speed, and caused by lose it is smaller.
Preferably, the step (3), wherein the specific additional amount of enzyme is laccase 0.24-0.36g/L, alkali protease
0.65-0.85g/L, Pullulanase 0.18-0.22g/L.This additional amount is relatively mild, and the albumen in algae can be made to obtain abundant enzyme
Solution, and not will cause material waste.
Preferably, the alkali protease is bacillus alkaline protease.Subtilopeptidase A is bacillus
Extracellular alkali protease secreted by bacterium belongs to serine protein hydrolase class, preferable to the protein breakdown effect in algae.
Preferably, the step (4), wherein the specific additional amount of pepsin is 0.60-0.86g/L.This additional amount compared with
To be moderate, the albumen in algae can be made sufficiently to be digested, and not will cause material waste.
Preferably, the ultrafiltration in the step (5), is surpassed with the ultrafiltration membrane that can intercept 10000 dalton of molecular weight
Filter.The ultrafiltration membrane of this specification carries out ultrafiltration, can sufficiently remove impurity, leave small-molecular peptides, and have higher recovery rate.
Preferably, the drying in the step (6) is in vacuum drier, and control temperature 45 C carries out freeze-day with constant temperature.
Vacuum constant temperature drying has faster rate of drying, and will not impact to small-molecular peptides.
Preferably, the drying in step (6) is spray drying.The drying process of spray drying is very fast, can directly do
It is dry at powder more be suitable for industrialized production.
Compared with prior art, the technical effect of the invention is embodied in:
Algae is dissolved in ultrasonication after water by the present invention, overflows the beneficiating ingredients such as the albumen in algae, reuse laccase,
Alkali protease, Pullulanase, pepsin carry out alkali to broken mixed solution, acid alternately digests the albumen made in algae
It is sufficiently dissociated, then carries out drying after ultrafiltration, method of the invention possesses high small-molecular peptides recovery rate, greatly improves
Utilization by algae rate reduces material waste, and possesses faster extraction rate, is that one kind is efficient, simple, fast algae is small
Molecular peptide extracting method.
Specific embodiment
It is limited below with reference to specific embodiment technical solution of the present invention is further, but claimed
Range is not only limited to made description.
Embodiment 1
(1) the spirulina 10kg cleaned up is weighed, is dissolved with water, keeps algae evenly dispersed in water, controls solid-liquid ratio
40g/L is obtained；
(2) aqueous solution for being mixed with algae is subjected to ultrasonication with the frequency of 30KHz at 0-10 DEG C；
(3) the mixed solution ph after ultrasonication is adjusted to 9.5, laccase 0.30g/L, Bacillus subtilis alkaline is added thereto
Proteinase-10 .75g/L, Pullulanase 0.20g/L, stir evenly, and digest 2h at 45 DEG C, by the enzyme-deactivating in mixed solution；
(4) the mixed liquor tune ph to 6.3 by enzyme deactivation after living, is added 0.72g/L pepsin, digests at 42.5 DEG C
30min, by the enzyme-deactivating in mixed solution；
(5) mixed liquor of the enzyme deactivation after living is subjected to ultrafiltration with the ultrafiltration membrane that can intercept 10000 dalton of molecular weight；
(6) by the concentration after ultrafiltration, in vacuum drier, after control temperature 45 C progress freeze-day with constant temperature to obtain the final product.
Embodiment 2
(1) the spirulina 10kg cleaned up is weighed, is dissolved with water, keeps algae evenly dispersed in water, controls solid-liquid ratio
20g/L is obtained；
(2) aqueous solution for being mixed with algae is subjected to ultrasonication with the frequency of 20KHz at 0-10 DEG C；
(3) the mixed solution ph after ultrasonication is adjusted to 9.2, laccase 0.24g/L, Bacillus subtilis alkaline is added thereto
Proteinase-10 .65g/L, Pullulanase 0.18g/L, stir evenly, and digest 1h at 55 DEG C, by the enzyme-deactivating in mixed solution；
(4) the mixed liquor tune ph to 6.6 by enzyme deactivation after living, is added 0.60g/L pepsin, digests 20min at 45 DEG C,
By the enzyme-deactivating in mixed solution；
(5) mixed liquor of the enzyme deactivation after living is subjected to ultrafiltration with the ultrafiltration membrane that can intercept 10000 dalton of molecular weight；
(6) by the concentration after ultrafiltration, in vacuum drier, after control temperature 45 C progress freeze-day with constant temperature to obtain the final product.
Embodiment 3
(1) spirulina cleaned up is weighed, is dissolved with water, keeps algae evenly dispersed in water, controls solid-liquid ratio 60g/L
It obtains；
(2) aqueous solution for being mixed with algae is subjected to ultrasonication with the frequency of 40KHz at 0-10 DEG C；
(3) the mixed solution ph after ultrasonication is adjusted to 9.8, laccase 0.36g/L, Bacillus subtilis alkaline is added thereto
Proteinase-10 .85g/L, Pullulanase 0.22g/L, stir evenly, and digest 4h at 35 DEG C, by the enzyme-deactivating in mixed solution；
(4) the mixed liquor tune ph to 6.1 by enzyme deactivation after living, is added 0.86g/L pepsin, digests 40min at 40 DEG C,
By the enzyme-deactivating in mixed solution；
(5) mixed liquor of the enzyme deactivation after living is subjected to ultrafiltration with the ultrafiltration membrane that can intercept 10000 dalton of molecular weight；
(6) by the concentration after ultrafiltration, in vacuum drier, after control temperature 45 C progress freeze-day with constant temperature to obtain the final product.
Embodiment 4
(1) the spirulina 10kg cleaned up is weighed, is dissolved with water, keeps algae evenly dispersed in water, controls solid-liquid ratio
40g/L is obtained；
(2) aqueous solution for being mixed with algae is subjected to ultrasonication with the frequency of 30KHz at 0-10 DEG C；
(3) the mixed solution ph after ultrasonication is adjusted to 9.5, laccase 0.30g/L, elastoser is added thereto
0.75g/L, Pullulanase 0.20g/L, stir evenly, and digest 2h at 45 DEG C, by the enzyme-deactivating in mixed solution；
(4) the mixed liquor tune ph to 6.3 by enzyme deactivation after living, is added 0.72g/L chymotrypsin, digests at 42.5 DEG C
30min, by the enzyme-deactivating in mixed solution；
(5) mixed liquor of the enzyme deactivation after living is subjected to ultrafiltration with the ultrafiltration membrane that can intercept 10000 dalton of molecular weight；
(6) by the concentration after ultrafiltration, in vacuum drier, after control temperature 45 C progress freeze-day with constant temperature to obtain the final product.
Embodiment 5
(1) the spirulina 10kg cleaned up is weighed, is dissolved with water, keeps algae evenly dispersed in water, controls solid-liquid ratio
40g/L is obtained；
(2) aqueous solution for being mixed with algae is subjected to ultrasonication with the frequency of 30KHz at 0-10 DEG C；
(3) the mixed solution ph after ultrasonication is adjusted to 9.5, laccase 0.30g/L, Bacillus subtilis alkaline is added thereto
Proteinase-10 .75g/L, Pullulanase 0.20g/L, stir evenly, and digest 2h at 45 DEG C, by the enzyme-deactivating in mixed solution；
(4) the mixed liquor tune ph to 6.3 by enzyme deactivation after living, is added 0.72g/L pepsin, digests at 42.5 DEG C
30min, by the enzyme-deactivating in mixed solution；
(5) mixed liquor of the enzyme deactivation after living is subjected to ultrafiltration with the ultrafiltration membrane that can intercept 10000 dalton of molecular weight；
(6) it by the concentration after ultrafiltration, is spray-dried to obtain the final product.
Comparative example 1
It is carried out according to the embodiment 1 of patent application CN201210375126.8.
Embodiment 1-5 and comparative example 1 are compared:
| Small-molecular peptides recovery rate (%) | Gained small-molecular peptides purity (%) | Extraction time (h) | |
| Embodiment 1 | 46.8% | 98.6 | 3h |
| Embodiment 2 | 44.3% | 97.8 | 2h |
| Embodiment 3 | 47.2% | 98.2 | 5h |
| Embodiment 4 | 47.6% | 98.5 | 3h |
| Embodiment 5 | 45.0% | 98.8 | 3h |
| Comparative example 1 | 14.8% | 90.6 | 3h |
By upper data it is found that method of the invention possesses high small-molecular peptides recovery rate, the benefit of algae is greatly improved
With rate, reduce material waste, and possess faster extraction rate, is that efficient, the simple, fast algae small-molecular peptides of one kind mention
Take method.
Finally it is pointed out that above embodiments are only the more representational examples of the present invention.Obviously, technology of the invention
Scheme is not limited to above-described embodiment, and acceptable there are many deformations.Those skilled in the art can be from disclosed by the invention
All deformations that content is directly exported or associated, are considered as protection scope of the present invention.
Claims (9)
1. a kind of algae small-molecular peptides extracting method, which comprises the steps of:
(1) algae cleaned up is weighed, is dissolved with water, keeps algae evenly dispersed in water, obtains；
(2) aqueous solution for being mixed with algae is subjected to ultrasonication；
(3) the mixed solution ph after ultrasonication is adjusted to 9.2-9.8, laccase, alkali protease, Propiram is added thereto
Enzyme stirs evenly, and digests 1-4h at 35-55 DEG C, by the enzyme-deactivating in mixed solution；
(4) the mixed liquor tune ph to 6.1-6.6 by enzyme deactivation after living, is added pepsin, digests 20-40min at 40-45 DEG C,
By the enzyme-deactivating in mixed solution；
(5) mixed liquor of the enzyme deactivation after living is subjected to ultrafiltration；
(6) by after ultrafiltration concentration, it is dry after to obtain the final product.
2. algae small-molecular peptides extracting method according to claim 1, which is characterized in that the step (1) controls feed liquid
Compare 20-60g/L.
3. algae small-molecular peptides extracting method according to claim 1, which is characterized in that the ultrasound of the step (2) is broken
It is broken, it is to carry out ultrasonication with the frequency of 20-40KHz at 0-10 DEG C.
4. algae small-molecular peptides extracting method according to claim 1, which is characterized in that the step (3), wherein enzyme
Specific additional amount is laccase 0.24-0.36g/L, alkali protease 0.65-0.85g/L, Pullulanase 0.18-0.22g/L.
5. algae small-molecular peptides extracting method according to claim 1 or 4, which is characterized in that the alkali protease is
Bacillus alkaline protease.
6. algae small-molecular peptides extracting method according to claim 1, which is characterized in that the step (4), wherein stomach egg
The specific additional amount of white enzyme is 0.60-0.86g/L.
7. algae small-molecular peptides extracting method according to claim 1, which is characterized in that the ultrafiltration in the step (5),
It is to carry out ultrafiltration with the ultrafiltration membrane that can intercept 10000 dalton of molecular weight.
8. algae small-molecular peptides extracting method according to claim 1, which is characterized in that the drying in the step (6),
It is in vacuum drier, control temperature 45 C carries out freeze-day with constant temperature.
9. algae small-molecular peptides extracting method according to claim 1, which is characterized in that the drying in the step (6),
It is spray drying.
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2019
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Application publication date: 20190809 |