CN109371088A - A kind of preparation method of sea cucumber active peptide - Google Patents
A kind of preparation method of sea cucumber active peptide Download PDFInfo
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Abstract
The present invention relates to technical field of polypeptide, specifically disclose a kind of preparation method of sea cucumber active peptide.The preparation method includes the following steps: to choose new fresh sea cucumber, obtains sea cucumber slurries through pulverization process；Collagenase, white gourd prolease is added, enzymatic hydrolysis obtains enzymolysis liquid；Enzymolysis liquid obtains sea cucumber active peptide through filtering, centrifugal degreasing, nanofiltration, desalination and concentration, deionizing purification, freeze-drying.The preparation method of sea cucumber active peptide provided by the invention, further increase degree of hydrolysis, keep hydrolysis more thorough, improve enzymolysis efficiency, high-molecular polypeptide is set to be hydrolyzed to low molecular active peptide, through separation, purifying and freeze-drying, obtain high concentration, high activity and the low molecular weight convenient for absorption of human body that molecular weight homogeneity is good biologically active peptide.
Description
Technical field
The present invention relates to technical field of polypeptide more particularly to a kind of preparation methods of sea cucumber active peptide.
Background technique
Sea cucumber is the ocean echinoderm to 8000 meters by the sea that lives, the history away from modern existing more than 600,000,000 years, sea cucumber with
Seabed algae and planktonic organism are food.Sea cucumber whole body covers with corn, is widely distributed in each ocean in the world.South China Sea bank type compared with
More, there are about more than 20 sea cucumbers can be edible.The same ginseng of sea cucumber, bird's nest, shark's fin are eponymous, are one of big treasures in the world eight.Sea cucumber
It is not only precious food and rare medicinal material.According to being recorded in supplementary Amplifications of the Compendium of Materia Medica: sea cucumber, sweet-salty, kidney tonifying, strengthening the essence
Marrow, takes the photograph urine, and impotence, warm-natured benefit, sufficient enemy's ginseng, therefore named sea cucumber are treated in establishing-Yang.Protein (90%) rich in sea cucumber body,
Fat, vitamin, saponin(e, polysaccharide isoreactivity ingredient, cholesterol level is low, health value with higher.Therefore, sea cucumber is out
The good source of biologically active peptide is sent out, they not only have a special physiological active functions, and abundance, safe and nontoxic
Side effect.
Currently, for sea cucumber active peptide extract enzymolysis protein type it is very much, by its source be broadly divided into animal origin,
Plant origin and microbe-derived three classes.Animal protease is mainly derived from the blood and alimentary canal of animal, mainly there is tryptose
Enzyme, chymotrypsin, pepsin, fibrin ferment, hyaluronidase etc..Plant rennet be mainly derived from plant organ and
Tissue, mainly there is chymopapain, papain, ficin, bromelain etc..Microbial protease master
To derive from various mycoprotein, such as subtilopeptidase A in bacterium, black mold and aspergillus oryzae in mould, Saccharomyces protein enzyme and
Unwrapping wire mycoproteinase etc..
Single enzymolysis or double-enzyme hydrolysis are generally used in existing enzymolysis method of sea cucumber, since enzyme is to the specificity of substrate,
The polypeptide yield that aminosal obtains is not very high, and the molecular weight homogeneity of obtained polypeptide is poor, it is difficult to be obtained highly concentrated
It spends, the biologically active peptide of the low molecular weight convenient for absorption of human body of high activity.
Summary of the invention
Low for protein hydrolysis degree in the prior art, polypeptide yield height and the molecular weight homogeneity difference of gained polypeptide etc. are asked
Topic, the present invention provide a kind of preparation method of sea cucumber active peptide.
To achieve the above object of the invention, the embodiment of the present invention uses the following technical solution:
A kind of preparation method of sea cucumber active peptide, includes the following steps:
(1) new fresh sea cucumber is chosen, internal organ are taken out and is cleaned, wall of sea cucumber Stichopus japonicus and sea cucumber intestine are obtained, carries out pulverization process, is added
The 7-8 times of purified water stirring of sea cucumber weight, obtains sea cucumber slurries；
(2) Collagenase is added into the sea cucumber slurries, is warming up to 50-60 DEG C, carry out enzymolysis processing 0.5h-
1.0h, is added white gourd prolease, heat preservation, and enzymolysis processing 2.5-3.5h heats up and carries out inactivation treatment to enzyme, obtains enzymolysis liquid；
(3) enzymolysis liquid is filtered processing, obtains filtrate, filtrate carries out centrifugal degreasing, obtains centrifugate；
(4) centrifugate obtains concentrate through nanofiltration, desalination and concentration；
(5) concentrate obtains sea cucumber active peptide through deionizing purification, freeze-drying.
Further, the Collagenase additional amount is the 1.5%-3% of sea cucumber weight, with generated in sea cucumber intestine from
Lyase synergistic effect, preliminary hydrolysis is carried out to sea cucumber albumen.
Further, the white gourd prolease additional amount is the 0.8%-1.5% of sea cucumber weight, white gourd prolease activity
By force, stability is high, helps to improve degree of hydrolysis, acts synergistically with Collagenase, further polypeptide is made to be hydrolyzed to low molecule
Active peptide, and be conducive to improve active peptide taste and flavor.
Further, the inactivation treatment temperature is 85-95 DEG C, time 10-15min.
Further, in step (3), the enzymolysis liquid is filtered processing using sleeping gong centrifuge, and the filtrate uses
Three-phase centrifuge carries out centrifugal degreasing processing.
Further, in step (4), the centrifugate uses aperture to carry out nanofiltration processing for the nanofiltration membrane of 0.5nm, penetrates
Liquid uses aperture to carry out desalination and concentration for the nanofiltration membrane of 0.2nm, and different points are separated using the nanofiltration membrane of different pore size
The polypeptide of son amount and removing single amino acids and salinity, removing are greater than the peptide of 500 dalton, make dipeptides to pentapeptide content by
57% is increased to 99%, obtains the biologically active peptide of high concentration, the low molecular weight convenient for absorption of human body of high activity.
Further, in step (5), the freeze-drying process method are as follows: in -45 DEG C~-40 DEG C pre-freeze 1.25-
1.35h is -42 DEG C~-20 DEG C in temperature, under the conditions of vacuum degree is 0.12mbar~0.15mbar, lyophilization 7.8-8.3h,
It is again -30 DEG C -10 DEG C in temperature, under the conditions of vacuum degree is 0.15mbar~0.25mbar, parsing-desiccation 2.5-3.5h.Distillation is dry
The dry Free water mainly in material, parsing-desiccation are mainly removed with the stronger absorption water of solid bond, the sea made
Ginseng active peptide instant capacity is good, nutriment loss is few, improves the quality of active peptide product.
Compared with the existing technology, the preparation method of sea cucumber active peptide provided by the invention is with wall of sea cucumber Stichopus japonicus and sea cucumber intestine
Main substrate makes the autolytic enzyme generated in sea cucumber body and Collagenase act synergistically, improves protein hydrolysis degree, carries out to albumen
Initial hydrolysis keeps molecular weight of albumen digestion uniform, and can save original active material in sea cucumber to the maximum extent；Add the winter
Melon protease and autolytic enzyme and Collagenase collective effect increase restriction enzyme site, further increase degree of hydrolysis, make hydrolysis more
Thoroughly, enzymolysis efficiency is improved, so that high-molecular polypeptide is hydrolyzed to low molecular active peptide, through separation, purifying and freeze-drying, obtains highly concentrated
Degree, high activity and the low molecular weight convenient for absorption of human body that molecular weight homogeneity is good biologically active peptide.
The sea cucumber active peptide that is prepared the present invention also provides above-mentioned preparation method and its preparing cosmetics, food
Product, health food, the application in drug.
Sea cucumber active peptide provided by the invention, molecular weight homogeneity is good, and bioactivity is high, has no toxic side effect, molecular weight phase
To lower, it will not cause as the allergic reaction caused by human body of protein or macromolecule peptide, it is easier to be digested, fit
It closes and is widely preparing the application of the fields such as cosmetics, food, health food, drug.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Embodiment 1
A kind of preparation method of sea cucumber active peptide, includes the following steps:
(1) choose new fresh sea cucumber 10kg, take out internal organ and simultaneously clean, obtain wall of sea cucumber Stichopus japonicus and sea cucumber intestine, using meat grinder into
Row crushes, and the stirring of 7L purified water is added, obtains sea cucumber slurries, interior to contain protein about 8.5kg；
(2) 150g Collagenase is added into the sea cucumber slurries, is warming up to 50 DEG C, carries out enzymolysis processing 1.0h, adds
Enter 80g white gourd prolease, 50 DEG C of heat preservations, enzymolysis processing 3.5h is warming up to 85 DEG C and carries out inactivation treatment 15min to enzyme, obtains enzyme
Liquid is solved, after measured, (loss amount includes the protein lost in pretreatment process and hydrolyzes containing polypeptide about 7.31kg in enzymolysis liquid
The free amino acid arrived)；
(3) enzymolysis liquid is filtered processing using sleeping gong centrifuge, obtains filtrate, filtrate uses three-phase centrifuge
Centrifugal degreasing is carried out, centrifugate is obtained；
(4) centrifugate first use aperture for 0.5nm nanofiltration membrane carry out nanofiltration processing, permeate use aperture for
The nanofiltration membrane of 0.2nm carries out desalination and concentration, obtains concentrate；
(5) concentrate in step (4) is subjected to deionizing purification with ion filter, is further concentrated with dual-effect concentrator
It afterwards, is -20 DEG C in temperature in -45 DEG C of pre-freeze 1.25h, under the conditions of vacuum degree is 0.12mbar, lyophilization 8h, then in temperature
It is -15 DEG C, under the conditions of vacuum degree is 0.15mbar, parsing-desiccation 3h obtains the sea cucumber active peptide that molecular weight is 180-500Da
6.05kg, polypeptide yield (active peptide obtained amount/total protein) are 71.2%.
Embodiment 2
A kind of preparation method of sea cucumber active peptide, includes the following steps:
(1) choose new fresh sea cucumber 10kg, take out internal organ and simultaneously clean, obtain wall of sea cucumber Stichopus japonicus and sea cucumber intestine, using meat grinder into
Row crushes, and the stirring of 75L purified water is added, obtains sea cucumber slurries, interior to contain protein about 8.7kg；
(2) 200g Collagenase is added into the sea cucumber slurries, is warming up to 55 DEG C, carries out enzymolysis processing 0.8h, adds
Enter 120g white gourd prolease, 55 DEG C of heat preservations, enzymolysis processing 3h is warming up to 95 DEG C and carries out inactivation treatment 10min to enzyme, digested
Liquid, after measured, the about 7.48kg containing polypeptide in enzymolysis liquid；
(3) enzymolysis liquid is filtered processing using sleeping gong centrifuge, obtains filtrate, filtrate uses three-phase centrifuge
Centrifugal degreasing is carried out, centrifugate is obtained；
(4) centrifugate first use aperture for 0.5nm nanofiltration membrane carry out nanofiltration processing, permeate use aperture for
The nanofiltration membrane of 0.2nm carries out desalination and concentration, obtains concentrate；
(5) concentrate in step (4) is subjected to deionizing purification with ion filter, is further concentrated with dual-effect concentrator
It afterwards, is -42 DEG C in temperature in -45 DEG C of pre-freeze 1.35h, under the conditions of vacuum degree is 0.12mbar, lyophilization 7.8h, then in temperature
Degree is -30 DEG C, and under the conditions of vacuum degree is 0.15mbar, parsing-desiccation 2.5h obtains the sea cucumber activity that molecular weight is 180-500Da
Peptide 6.39kg, polypeptide yield 73.4%.
Embodiment 3
A kind of preparation method of sea cucumber active peptide, includes the following steps:
(1) choose new fresh sea cucumber 10kg, take out internal organ and simultaneously clean, obtain wall of sea cucumber Stichopus japonicus and sea cucumber intestine, using meat grinder into
Row crushes, and the stirring of 80L purified water is added, obtains sea cucumber slurries and has included protein about 9.1kg；
(2) 300g Collagenase is added into the sea cucumber slurries, is warming up to 60 DEG C, carries out enzymolysis processing 0.5h, adds
Enter 150g white gourd prolease, 60 DEG C of heat preservations, enzymolysis processing 2.5h is warming up to 90 DEG C and carries out inactivation treatment 13min to enzyme, obtains enzyme
Solve liquid, after measured, the about 8.15kg containing polypeptide in enzymolysis liquid；
(3) enzymolysis liquid is filtered processing using sleeping gong centrifuge, obtains filtrate, filtrate uses three-phase centrifuge
Centrifugal degreasing is carried out, centrifugate is obtained；
(4) centrifugate first use aperture for 0.5nm nanofiltration membrane carry out nanofiltration processing, permeate use aperture for
The nanofiltration membrane of 0.2nm carries out desalination and concentration, obtains concentrate；
(5) concentrate in step (4) is subjected to deionizing purification with ion filter, is further concentrated with dual-effect concentrator
It afterwards, is -25 DEG C in temperature in -45 DEG C of pre-freeze 1.3h, under the conditions of vacuum degree is 0.15mbar, lyophilization 8.3h, then in temperature
It is -10 DEG C, under the conditions of vacuum degree is 0.25mbar, parsing-desiccation 3.5h obtains the sea cucumber active peptide that molecular weight is 180-500Da
6.86kg, polypeptide yield 75.4%.
Technical solution in order to better illustrate the present invention is done by comparative example and the embodiment of the present invention into one further below
The comparison of step.
Comparative example 1
Collagenase in embodiment 1 is replaced with into papain, other operations are same as Example 1, obtain sea
Join active peptide, polypeptide yield 47.3%.
Comparative example 2
White gourd prolease in embodiment 1 is replaced with into bromelain, other operations are same as Example 1, obtain sea
Join active peptide, polypeptide yield 49.5%.
The preparation method for the sea cucumber active peptide that embodiment provides in order to better illustrate the present invention, below by embodiment 1-3
And enzymolysis liquid degree of being hydrolyzed obtained in comparative example 1-2 preparation process measurement and to obtained centrifugate carry out molecular weight
Measurement.
Degree of hydrolysis measurement
TCA (trichloroacetic acid) method: principle is that protein is positively charged in acid condition, in conjunction with the acid group of trichloroacetic acid
Form precipitating.
5mL enzymolysis liquid is taken to be added to the TCA of 5mL10%, mixing is shaken, 4000r/min centrifugation 20min, in enzymolysis liquid not
The protein of hydrolysis and the peptide fragment of macromolecule are precipitated by TCA, and supernatant is gone to be diluted in the measurement range of standard curve
The measurement of soluble polypeptide is carried out, the content of soluble polypeptide uses micro-Kjeldahl respectively in total protein content and supernatant
It is measured with Forint phenol method.
Degree of hydrolysis (degree of hydrolysis, DH) %=(N2-N1)/(N0-N1)
N0For the total protein in sea cucumber albumen, N1For the TCA soluble polypeptide before enzyme digestion reaction in sea cucumber albumen, N2For enzyme
Solve the TCA soluble polypeptide in liquid.
The degree of hydrolysis test result of enzymolysis liquid is as shown in table 1 in embodiment and comparative example.
Table 1
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example 1 | Comparative example 2 | |
| Degree of hydrolysis, % | 89.7 | 90.6 | 92.4 | 72.3 | 76.4 |
Data can obtain in table, the preparation method of sea cucumber active peptide provided in an embodiment of the present invention, white gourd prolease and collagen
Protease and autolytic enzyme collective effect expand restriction enzyme site, further increase degree of hydrolysis, degree of hydrolysis is up to 90% or so, makes water
Solution is more thorough, improves enzymolysis efficiency, improves polypeptide yield.
The measurement of molecular weight
Detection method: molecular weight is measured by gel chromatography, is selected at the Tris-HCl eluent balance of 0.1mol/L
After managing SPhedexG-25 gel column, under the wavelength of 220nm, detectable concentration is the standard substance of 2mg/ml: reproducibility gluathione
Peptide (307Da), oxidative glutathione (612Da) oxytocin (1007Da) and glucagon (3485Da), according to reference substance
Matter molecular weight logarithm and elution volume are fitted to obtain regression equation: y=-38.15x+198.6 (R2=0.9932)；Wherein, y is to wash
Lift-off product, x are the logarithm of corresponding molecular weight.
Centrifugate obtained by each embodiment and comparative example is freeze-dried, freeze-dried powder is obtained, freeze-dried powder is respectively configured
It is 5mg/ml for concentration, sample introduction elutes respectively under the above conditions, brings appearance volume into regression equation standard curve, can obtain
Molecular weight analyte and its distribution determine the percentage contents of different molecular weight protein peptides, institute by peak area normalization method
The results are shown in Table 2.
Table 2
| ≥500Da | 180-500Da | < 180Da | |
| Embodiment 1 | 9.2% | 80.3% | 10.5% |
| Embodiment 2 | 9.3% | 81.7% | 9% |
| Embodiment 3 | 7.3% | 82.3% | 10.4% |
| Comparative example 1 | 24.6% | 65.4% | 10% |
| Comparative example 2 | 28.9% | 63.1% | 8% |
It can be obtained by above data, sea cucumber active peptide provided in an embodiment of the present invention, molecular weight is mainly distributed on 180-
500Da makes human body be easier to be absorbed and utilized, and can be widely used in cosmetics, food, health food and drug etc..
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modification, equivalent replacement or improvement etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (8)
1. a kind of preparation method of sea cucumber active peptide, characterized by the following steps:
(1) new fresh sea cucumber is chosen, internal organ are taken out and is cleaned, wall of sea cucumber Stichopus japonicus and sea cucumber intestine are obtained, carries out pulverization process, sea cucumber is added
7-8 times of purified water stirring of weight, obtains sea cucumber slurries；
(2) Collagenase is added into the sea cucumber slurries, is warming up to 50-60 DEG C, carry out enzymolysis processing 0.5h-1.0h, add
Enter white gourd prolease, keep the temperature, enzymolysis processing 2.5-3.5h, heating carries out inactivation treatment to enzyme, obtains enzymolysis liquid；
(3) enzymolysis liquid is filtered processing, obtains filtrate, filtrate carries out centrifugal degreasing, obtains centrifugate；
(4) centrifugate obtains concentrate through nanofiltration, desalination and concentration；
(5) concentrate obtains sea cucumber active peptide through deionizing purification, freeze-drying.
2. the preparation method of sea cucumber active peptide as described in claim 1, it is characterised in that: the Collagenase additional amount is
The 1.5%-3% of sea cucumber weight.
3. the preparation method of sea cucumber active peptide as described in claim 1, it is characterised in that: the white gourd prolease additional amount is
The 0.8%-1.5% of sea cucumber weight.
4. the preparation method of sea cucumber active peptide as described in claim 1, it is characterised in that: the inactivation treatment temperature is 85-
95 DEG C, time 10-15min.
5. the preparation method of sea cucumber active peptide as described in claim 1, which is characterized in that in step (3), the enzymolysis liquid is adopted
It is filtered processing with sleeping gong centrifuge, the filtrate carries out centrifugal degreasing processing using three-phase centrifuge.
6. the preparation method of sea cucumber active peptide as described in claim 1, it is characterised in that: in step (4), the centrifugate is adopted
Nanofiltration processing is carried out with the nanofiltration membrane that aperture is 0.5nm, permeate uses aperture to carry out desalination and concentration for the nanofiltration membrane of 0.2nm
Processing.
7. the preparation method of sea cucumber active peptide as described in claim 1, it is characterised in that: in step (5), the freeze-drying
Processing method are as follows: it is -42 DEG C~-20 DEG C in temperature in -45 DEG C~-40 DEG C pre-freeze 1.25-1.35h, vacuum degree 0.12mbar
Under the conditions of~0.15mbar, lyophilization 7.8-8.3h, then temperature be -30 DEG C -10 DEG C, vacuum degree be 0.15mbar~
Under the conditions of 0.25mbar, parsing-desiccation 2.5-3.5h.
8. the sea cucumber active peptide that the described in any item preparation methods of claim 1-7 are prepared.
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| CN110604209A (en) * | 2019-10-23 | 2019-12-24 | 青岛琅琊龙湾海参股份有限公司 | Preparation method of sea cucumber bioactive peptide nanoparticles |
| CN111876275A (en) * | 2020-06-19 | 2020-11-03 | 齐鲁工业大学 | Brewing method of sea cucumber beer |
| CN113801907A (en) * | 2020-06-11 | 2021-12-17 | 中国热带农业科学院农产品加工研究所 | Preparation method of radix adenophorae polypeptide |
| CN114507703A (en) * | 2022-03-10 | 2022-05-17 | 青岛浩然海洋科技有限公司 | Preparation process of sea cucumber peptide |
| CN115918770A (en) * | 2022-10-20 | 2023-04-07 | 山东海龙元生物科技有限公司 | Preparation method of marine organism health-preserving health-care peptide preparation |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110604209A (en) * | 2019-10-23 | 2019-12-24 | 青岛琅琊龙湾海参股份有限公司 | Preparation method of sea cucumber bioactive peptide nanoparticles |
| CN113801907A (en) * | 2020-06-11 | 2021-12-17 | 中国热带农业科学院农产品加工研究所 | Preparation method of radix adenophorae polypeptide |
| CN111876275A (en) * | 2020-06-19 | 2020-11-03 | 齐鲁工业大学 | Brewing method of sea cucumber beer |
| CN114507703A (en) * | 2022-03-10 | 2022-05-17 | 青岛浩然海洋科技有限公司 | Preparation process of sea cucumber peptide |
| CN115918770A (en) * | 2022-10-20 | 2023-04-07 | 山东海龙元生物科技有限公司 | Preparation method of marine organism health-preserving health-care peptide preparation |
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