CN108660177A - A kind of sea cucumber polypeptide - Google Patents
A kind of sea cucumber polypeptide Download PDFInfo
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- CN108660177A CN108660177A CN201810292047.8A CN201810292047A CN108660177A CN 108660177 A CN108660177 A CN 108660177A CN 201810292047 A CN201810292047 A CN 201810292047A CN 108660177 A CN108660177 A CN 108660177A
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- sea cucumber
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- enzymolysis
- cucumber polypeptide
- polypeptide
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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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- 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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- 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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
Abstract
The invention discloses a kind of sea cucumber polypeptide, preparation method includes：Pretreatment, fermentation, enzymolysis, ultrafiltration, sea cucumber is impregnated first and is homogenized after easing back, fermentation synergist is added and obtains fermentation substrate, it is inoculated in fermentation substrate and carries out fermented and cultured into aspergillus oryzae, S. cervisiae and obtain zymotic fluid through miillpore filter, neutral proteinase is added in zymotic fluid to be digested, enzymolysis liquid ultrafiltration after removal of impurities obtains sea cucumber polypeptide.It has the beneficial effect that：Sea cucumber polypeptide has four sections of different molecular weights, not only avoid the waste of active peptides in zymotic fluid, it can also more refine, study to differentiated the biological activity of segment polypeptide between each molecular weight area, sea cucumber polypeptide has effects that remove the physiology such as free radical, anti-oxidative damage, hypoglycemic, improvement sugar tolerance, the preparation process of sea cucumber polypeptide is simple, yield and purity are higher, and fermentation substantially increases the yield and yield of sea cucumber polypeptide with enzymolysis process.
Description
Technical field
The present invention relates to sea cucumber field of deep, more particularly, to a kind of sea cucumber polypeptide.
Technical background
Sea cucumber belongs to Holothuroidea（Holothuroidea）, it is the ocean echinoderm to 8000 meters by the sea that lives, away from the present
Has more than 600,000,000 years history, sea cucumber is food with seabed algae and planktonic organism.Sea cucumber whole body covers with corn, is widely distributed in the world
In each ocean.South China Sea bank type is more, and there are about more than 20 kind sea cucumbers can be edible.The same ginseng of sea cucumber, bird's nest, shark's fin are neat
Name, is one of eight big treasure of the world.Sea cucumber is not only precious food and rare medicinal material.According to《A Supplement to the Compendium of Materia Medica》In
It records：Sea cucumber, sweet-salty, kidney tonifying, benefiting quintessence take the photograph urine, and impotence, warm-natured benefit, sufficient enemy's ginseng, therefore named sea cucumber are treated in establishing-Yang.Sea cucumber
With improve memory, delay sexual gland aging, prevent artery sclerosis and it is antitumor the effects that.As sea cucumber is worth the general of knowledge
And sea cucumber progresses into common people's dining table.Living environment determines sea cucumber quality.Sea cucumber can adjust body moisture balance, be suitable for pregnant
The Ms of phase legs and feet edema.Dong Ze flagship stores sea cucumber dispelling fatigue, improves the immunity of the human body, the energy that enhancing human body resists the disease
Power, therefore be very suitable for often in the middle aged Ms of fatigue state and man, easy catching a cold, the elderly having a delicate constitution and children
Equal sub-health populations.Taurine, lysine in sea cucumber etc. in vegetable food almost without.The distinctive active material of sea cucumber
Holothurin has significant inhibiting effect, stichopus japonicus element A and B to can be used for treating fungi and T.mentagrophytes infection a variety of fungies, has aobvious
Anti-inflammatory, the osteogenic action write, especially has significant therapeutic effect to hepatitis, tuberculosis, diabetes, cardiovascular disease.
Invention content
The purpose of the present invention is to provide a kind of sea cucumber polypeptide, sea cucumber polypeptide has four sections of different molecular weights, has and removes
Free radical, anti-oxidative damage, it is hypoglycemic, improve the physiology effect such as sugar tolerance, the preparation process of sea cucumber polypeptide is simple, yield with
Purity is higher, and fermentation substantially increases the yield and yield of sea cucumber polypeptide with enzymolysis process.
Used microorganism and its bought enterprise are in the method for the present invention：Aspergillus oryzae, the micro- agriculture biotechnology in Baoding have
Limit company；S. cervisiae, Yantai Man Sen commerce and trade Co., Ltd.
The present invention is directed to the problem of being mentioned in background technology, and the technical solution taken is：A kind of sea cucumber polypeptide has four
The polypeptide of section different molecular weight, respectively T1（>10kDa）、T2（10~3kDa）、T3（3~1kDa）With T4（<1kDa）, sea cucumber
The essential amino acid composition of polypeptide is glycine and alanine, and sea cucumber polypeptide is to DPPH, OH and O2-It is removed with preferable
Effect, while sea cucumber polypeptide can significantly reduce the concentration of total cholesterol, triglycerides in serum and liver, can also promote
NIH/3T3 cell growths have significant facilitation to the secretion of NIH/3T3 cell collagen albumen, also have certain antioxygen
Change lesion capability, reduces blood glucose, improves the effect of sugar tolerance.
Preferably, the preparation method of sea cucumber polypeptide includes：Pretreatment, fermentation, enzymolysis, ultrafiltration, specifically include following step
Suddenly：
Pretreatment：First sea cucumber is cleaned with clear water, then impregnates 8~12h with clear water and eases back to sea cucumber, 3~5min is rinsed with flowing water
After dry, by solid-liquid ratio 1:10~12 homogenate, low temperature drying to water content are 35~45%, are added according to sea cucumber dry weight 0.2~0.4%
Enter fermentation synergist, is uniformly mixed up to fermentation substrate；Sea cucumber maceration can be made to silt, dirt, the part salinity inside sea cucumber
Exudation, is conducive to that it is further processed；
Fermentation：The aspergillus oryzae seed liquor of fermentation substrate quality 4~5%, 2~4% S. cervisiae seed liquor is taken to be inoculated with into fermentation
In matrix, 48~72h of fermented and cultured under 140~160r/min rotating speeds, 28~30 DEG C of temperature conditions；After fermentation, it will send out
Zymotic fluid boils 8~10min enzyme deactivations, and 8600~10000r/min centrifuges 15~30min, and supernatant crosses 0.2~0.25 μm of micropore filter
Film, filtrate are zymotic fluid；Under this fermentation condition, the sea cucumber glycogen rate of recovery and protein recovery respectively up to 85% and 90%,
After protein is hydrolyzed to polypeptide, become be easy to absorb, dissolubility is good, emulsibility is good, zymotic fluid with a variety of physiological activity,
The functional characteristic for improving protein improves nutrition and the utility value of protein, can be played using mixed fermentation more
Synergistic effect between kind of strain, can reduce the disadvantage of single bacterium fermentation, and there may be synbiosis, metabolite to occur between strain
Complementary effect, the unfavorable factor that intermediate product can be overcome excessively to be generated to antibacterial peptide, sea cucumber albumen prepare active peptides at
This is lower, and fermentation reaction is milder, and fermentation process is easily-controllable；
Enzymolysis：It is 75~80% by zymotic fluid low temperature drying to water content, adds 3000~4500U/g neutral proteinases, add
(R) -1 of neutral proteinase weight 1.5~2.3 ‰, 1 '-dinaphthalene -2,2 '-phenolic group diisopropyl oxygen titaniums, hydrolysis temperature be 55~
58 DEG C, enzymolysis pH value digest 1.5~2.0h under conditions of being 7.2~7.4, boiling 12~15min enzyme deactivations after enzymolysis obtains enzyme
Solve liquid；(R) -1,1 '-dinaphthalene -2,2 '-phenolic group diisopropyl oxygen titaniums can act on the metallic atom activated centre of protease, reduce
Energy barrier between albumen enzyme-to-substrate increases the reactivity between enzyme-to-substrate, to greatly improve the enzyme of protease
Solution activity, accelerates enzymolysis, so that the degree of hydrolysis of sea cucumber albumen is can reach 58% or more, improve the yield and yield of sea cucumber polypeptide,
With higher promotional value；
Ultrafiltration：Enzymolysis liquid is taken to sequentially add activated carbon, DA201-C macroporous resin adsorptions grease, impurity particle and desalination, it is rear to use
400~500 mesh sock filtrations remove adsorbent, add florisil silica combination plate filter be removed by filtration activated carbon with it is more
Excess oil fat obtains more clear enzymolysis liquid；It is fermented with the ultrafiltration membrane classified filtering that molecular cut off is 10KDa, 3KDa, 1kDa
Liquid obtains the polypeptide mixed liquor component T1 of four sections of different molecular weights（>10kDa）、T2（10~3kDa）、T3（3~1kDa）With T4
（<1kDa）, 48~60h is freeze-dried in the case where temperature is -10~-20 DEG C, vacuum degree is 50~100Pa up to four respectively after concentration
The sea cucumber polypeptide sterling of section different molecular weight；Enzymolysis liquid is subjected to ultrafiltration by three kinds of ultrafiltration membranes, obtains four sections of different molecular weights
Polypeptide mixed liquor, not only avoid the waste of active peptides in zymotic fluid, can also more refine, study to differentiated
The biological activity of segment polypeptide between each molecular weight area；The essential amino acid composition of sea cucumber polypeptide is glycine and alanine, sea
Join polypeptide to DPPH, OH and O2-With preferable elimination effect, while sea cucumber polypeptide can significantly reduce serum and liver
The concentration of middle total cholesterol, triglycerides can also promote NIH/3T3 cell growths, divide NIH/3T3 cell collagen albumen
Secreting has significant facilitation, also has effects that certain anti-oxidative damage ability, reduces blood glucose, improves sugar tolerance.
Preferably, the fermentation synergist in pre-treatment step includes the substance of following content：65~80% urea, 2~6%
α-naphthaleneacidsodium, 15~20% calcium oxide, 3~7% magnesium sulfate, 1~5% dimethyl phosphate, total amount 100%；In fermentation synergist
The α-naphthaleneacidsodium of special proportioning with dimethyl phosphate there is synergistic effect, the synergistic effect S. cervisiae can be promoted to enhance
Secretion to acetyl coenzyme A, and then promote its condensation reaction with oxaloacetic acid, accelerate the synthesis of citric acid, it can be significantly
The speed for improving S. cervisiae tricarboxylic acid cycle, accelerates the growth and breeding and fermentating metabolism of S. cervisiae, promotes ferment of making wine
Female bacterium is metabolized out more enzymes and is acted on fermentation substrate, and then improves the yield and yield of sea cucumber polypeptide.
Compared with the prior art, the advantages of the present invention are as follows：
1）The method of the present invention can obtain the polypeptide mixed liquor of four sections of different molecular weights, and it is more not only to avoid activity in zymotic fluid
The waste of peptide can also be refined more, study to differentiated the biological activity of segment polypeptide between each molecular weight area；
2）Synergistic effect between a variety of strains can be played using mixed fungus fermentation, reduce the disadvantage of single bacterium fermentation, it may between strain
Generation synbiosis, metabolite generation complementary effect, the unfavorable factor that intermediate product can be overcome excessively to be generated to antibacterial peptide,
Therefore be conducive to the yield of polypeptide and the raising of yield；
3）The α-naphthaleneacidsodium Yu dimethyl phosphate of special proportioning have synergistic effect in fermentation synergist when fermentation, can strengthen
S. cervisiae secretes acetyl coenzyme A, and then enhances its condensation reaction with oxaloacetic acid, accelerates the synthesis of citric acid, substantially
Degree improves the speed of S. cervisiae tricarboxylic acid cycle, accelerates the growth and breeding and fermentating metabolism of S. cervisiae, promotes to make wine
Metabolism of yeasts goes out more enzymes and is acted on fermentation substrate, and then improves the yield and yield of polypeptide；
4）When being digested to zymotic fluid, (R) -1,1 '-dinaphthalene -2,2 '-phenolic group diisopropyl oxygen titaniums can act on protease
Metallic atom activated centre reduces the energy barrier between protease and substrate protein white matter, increases between enzyme-to-substrate protein
Reactivity accelerate enzymolysis to greatly improve the enzymolysis activity of protease, the degree of hydrolysis of sea cucumber albumen made to can reach 58%
More than, there is higher promotional value.
Specific implementation mode
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of sea cucumber polypeptide, preparation method includes the following steps：
1）First sea cucumber is cleaned with clear water, then impregnates 8h with clear water and eases back to sea cucumber, is dried after rinsing 3min with flowing water, by feed liquid
Than 1:10 homogenate, low temperature drying to water content are 35%, are uniformly mixed up to fermentation substrate；2）Take the rice of fermentation substrate quality 4%
Aspergillus seed liquor, 2% S. cervisiae seed liquor be inoculated in fermentation substrate, under 140r/min rotating speeds, 28 DEG C of temperature conditions
Fermented and cultured 48h；After fermentation, zymotic fluid is boiled into 8min enzyme deactivations, 8600r/min centrifuges 15min, and supernatant crosses 0.2 μm
Miillpore filter, filtrate are zymotic fluid；3）It is 75% by zymotic fluid low temperature drying to water content, adds 3000U/g neutral proteins
Enzyme adds (R) -1 of neutral proteinase weight 1.5 ‰, 1 '-dinaphthalene -2, and 2 '-phenolic group diisopropyl oxygen titaniums are in hydrolysis temperature
55 DEG C, enzymolysis pH value be 7.2 under conditions of digest 1.5h, 12min enzyme deactivations are boiled after enzymolysis and obtain enzymolysis liquid；4）Take enzymolysis liquid
Activated carbon, DA201-C macroporous resin adsorptions grease, impurity particle and desalination are sequentially added, rear removed with 400 mesh sock filtrations is inhaled
It attached dose, adds florisil silica combination plate filter and is removed by filtration activated carbon and excess oil, obtain more clear enzymolysis
Liquid；With the ultrafiltration membrane classified filtering zymotic fluid that molecular cut off is 10KDa, 3KDa, 1kDa, the more of four sections of different molecular weights are obtained
Peptide mixed liquor component T1（>10kDa）、T2（10~3kDa）、T3（3~1kDa）With T4（<1kDa）, respectively in temperature after concentration
It is the sea cucumber polypeptide sterling that 48h is freeze-dried under 50Pa up to four sections of different molecular weights for -10 DEG C, vacuum degree, sea cucumber polypeptide
Comprehensive yield is 36.5%.
Embodiment 2：
A kind of sea cucumber polypeptide, the polypeptide with four sections of different molecular weights, respectively>10kDa, 10~3kDa, 3~1kDa with<
The essential amino acid composition of 1kDa, sea cucumber polypeptide are glycine and alanine, and sea cucumber polypeptide is to DPPH, OH and O2-With compared with
Good elimination effect, while sea cucumber polypeptide can significantly reduce the concentration of total cholesterol, triglycerides in serum and liver, may be used also
To promote NIH/3T3 cell growths, there is significant facilitation to the secretion of NIH/3T3 cell collagen albumen, also have certain
Anti-oxidative damage ability, reduce blood glucose, improve sugar tolerance the effect of.
The preparation method of sea cucumber polypeptide includes：Pretreatment, fermentation, enzymolysis, ultrafiltration, specifically include following steps：
1）Pretreatment：First sea cucumber is cleaned with clear water, then impregnates 12h with clear water and eases back to sea cucumber, is dried in the air after rinsing 5min with flowing water
It is dry, by solid-liquid ratio 1:12 homogenate, low temperature drying to water content are 45%, and fermentation synergist is added according to sea cucumber dry weight 0.4%, prepares
Fermentation synergist in fermentation medium step includes the substance of following content：70% urea, 4.8% α-naphthaleneacidsodium, 18% oxidation
Calcium, 6% magnesium sulfate, 1.2% dimethyl phosphate are uniformly mixed up to fermentation substrate；By sea cucumber maceration can make silt inside sea cucumber,
Dirt, the exudation of part salinity, are conducive to that it is further processed；
2）Fermentation：The aspergillus oryzae seed liquor of fermentation substrate quality 5%, 4% S. cervisiae seed liquor is taken to be inoculated with into fermentation substrate
In, the fermented and cultured 72h under 160r/min rotating speeds, 30 DEG C of temperature conditions；After fermentation, zymotic fluid is boiled into 10min enzyme deactivations,
10000r/min centrifuges 30min, and supernatant crosses 0.25 μm of miillpore filter, and filtrate is zymotic fluid；Under this fermentation condition, sea
Join the glycogen rate of recovery and protein recovery respectively up to 85% and 90%, after protein is hydrolyzed to polypeptide, become be easy to absorb,
Dissolubility is good, emulsibility is good, zymotic fluid with a variety of physiological activity, improves the functional characteristic of protein, improves albumen
The nutrition of matter and utility value can play the synergistic effect between a variety of strains using mixed fermentation, can reduce single bacterium hair
The disadvantage of ferment, there may be synbiosis, metabolite, and complementary effect occurs, intermediate product can be overcome excessively to fight between strain
The unfavorable factor that bacterium peptide generates, the cost that sea cucumber albumen prepares active peptides is lower, and fermentation reaction is milder, and fermentation process is easy
Control；
3）Enzymolysis：It is 80% by zymotic fluid low temperature drying to water content, adds 4500U/g neutral proteinases, add neutral protein
(R) -1 of enzyme weight 2.3 ‰, 1 '-dinaphthalene -2,2 '-phenolic group diisopropyl oxygen titaniums, hydrolysis temperature is 58 DEG C, enzymolysis pH value is 7.4
Under conditions of digest 2.0h, 15min enzyme deactivations are boiled after enzymolysis and obtain enzymolysis liquid；(R) -1,1 '-dinaphthalene -2,2 '-phenolic group two is different
Third oxygen titanium can act on the metallic atom activated centre of protease, reduce the energy barrier between albumen enzyme-to-substrate, increase
Reactivity between enzyme-to-substrate accelerates enzymolysis, makes the hydrolysis of sea cucumber albumen to greatly improve the enzymolysis activity of protease
Degree can reach 58% or more, improve the yield and yield of sea cucumber polypeptide, have higher promotional value；
4）Ultrafiltration：Enzymolysis liquid is taken to sequentially add activated carbon, DA201-C macroporous resin adsorptions grease, impurity particle and desalination, it is rear to use
500 mesh sock filtrations remove adsorbent, add florisil silica combination plate filter and are removed by filtration activated carbon and extra oil
Fat obtains more clear enzymolysis liquid；With the ultrafiltration membrane classified filtering zymotic fluid that molecular cut off is 10KDa, 3KDa, 1kDa, obtain
To the polypeptide mixed liquor component T1 of four sections of different molecular weights（>10kDa）、T2（10~3kDa）、T3（3~1kDa）With T4（<
1kDa）, 60h is freeze-dried in the case where temperature is -20 DEG C, vacuum degree is 100Pa up to four sections of different molecular weights respectively after concentration
The synthesis yield of sea cucumber polypeptide sterling, sea cucumber polypeptide is 40.3%；Enzymolysis liquid is subjected to ultrafiltration by three kinds of ultrafiltration membranes, obtains four
The polypeptide mixed liquor of section different molecular weight, not only avoids the waste of active peptides in zymotic fluid, can also more refine,
Study to differentiated the biological activity of segment polypeptide between each molecular weight area；The essential amino acid composition of sea cucumber polypeptide is glycine
And alanine, sea cucumber polypeptide is to DPPH, OH and O2-With preferable elimination effect, while sea cucumber polypeptide can significantly reduce
The concentration of total cholesterol, triglycerides in serum and liver, can also promote NIH/3T3 cell growths, to NIH/3T3 cell glues
The secretion of former albumen has significant facilitation, also has certain anti-oxidative damage ability, reduces blood glucose, improves sugar tolerance
Effect.
Embodiment 3：
A kind of sea cucumber polypeptide, the polypeptide with four sections of different molecular weights, respectively T1（>10kDa）、T2（10~3kDa）、T3
（3~1kDa）With T4（<1kDa）, the essential amino acid composition of sea cucumber polypeptide is glycine and alanine, sea cucumber polypeptide pair
DPPH, OH and O2-With preferable elimination effect, while sea cucumber polypeptide can significantly reduce total courage in serum and liver and consolidate
The concentration of alcohol, triglycerides can also promote NIH/3T3 cell growths, have significantly to the secretion of NIH/3T3 cell collagen albumen
Facilitation, also have effects that certain anti-oxidative damage ability, reduce blood glucose, improve sugar tolerance.
The preparation method of sea cucumber polypeptide includes：Pretreatment, fermentation, enzymolysis, ultrafiltration, specifically include following steps：
Pretreatment：First sea cucumber is cleaned with clear water, then impregnates 10h with clear water and eases back to sea cucumber, is dried after rinsing 5min with flowing water,
By solid-liquid ratio 1:10 homogenate, low temperature drying to water content are 42%, fermentation synergist are added according to sea cucumber dry weight 0.3%, mixing is equal
Even fermentation substrate to obtain the final product；Silt, dirt, part salinity inside sea cucumber can be made to ooze out sea cucumber maceration, be conducive to carry out it
Further processing；
Fermentation：The aspergillus oryzae seed liquor of fermentation substrate quality 4%, 4% S. cervisiae seed liquor is taken to be inoculated in fermentation substrate,
The fermented and cultured 60h under 140r/min rotating speeds, 28 DEG C of temperature conditions；After fermentation, zymotic fluid is boiled into 10min enzyme deactivations,
9000r/min centrifuges 25min, and supernatant crosses 0.2 μm of miillpore filter, and filtrate is zymotic fluid；Under this fermentation condition, sea cucumber
The glycogen rate of recovery and protein recovery are respectively up to 85% and 90%, after protein is hydrolyzed to polypeptide, becomes and are easy to absorb, are molten
Solution property is good, emulsibility is good, zymotic fluid with a variety of physiological activity, improves the functional characteristic of protein, improves protein
Nutrition and utility value, can play the synergistic effect between a variety of strains using mixed fermentation, can reduce single bacterium fermentation
Disadvantage, there may be synbiosis, metabolite, and complementary effect occurs, intermediate product can be overcome excessively to antibacterial between strain
The unfavorable factor that peptide generates, the cost that sea cucumber albumen prepares active peptides is lower, and fermentation reaction is milder, and fermentation process is easily-controllable；
Enzymolysis：It is 78% by zymotic fluid low temperature drying to water content, adds 4000U/g neutral proteinases, add neutral proteinase
(R) -1 of weight 2 ‰, 1 '-dinaphthalene -2,2 '-phenolic group diisopropyl oxygen titaniums, in the item that hydrolysis temperature is 56 DEG C, enzymolysis pH value is 7.3
2h is digested under part, and 15min enzyme deactivations are boiled after enzymolysis and obtain enzymolysis liquid；(R) -1,1 '-dinaphthalene -2,2 '-phenolic group diisopropyl oxygen titanium
The metallic atom activated centre of protease is can act on, the energy barrier between albumen enzyme-to-substrate is reduced, increases enzyme and bottom
Reactivity between object accelerates enzymolysis, keeps the degree of hydrolysis of sea cucumber albumen reachable to greatly improve the enzymolysis activity of protease
To 58% or more, the yield and yield of sea cucumber polypeptide are improved, there is higher promotional value；
Ultrafiltration：Enzymolysis liquid is taken to sequentially add activated carbon, DA201-C macroporous resin adsorptions grease, impurity particle and desalination, it is rear to use
450 mesh sock filtrations remove adsorbent, add florisil silica combination plate filter and are removed by filtration activated carbon and extra oil
Fat obtains more clear enzymolysis liquid；With the ultrafiltration membrane classified filtering zymotic fluid that molecular cut off is 10KDa, 3KDa, 1kDa, obtain
To the polypeptide mixed liquor component T1 of four sections of different molecular weights（>10kDa）、T2（10~3kDa）、T3（3~1kDa）With T4（<
1kDa）, 48h is freeze-dried after concentration in the case where temperature is -15 DEG C, vacuum degree is 60Pa respectively up to the sea of four sections of different molecular weights
Join polypeptide sterling, the synthesis yield of sea cucumber polypeptide is 42.6%；Enzymolysis liquid is subjected to ultrafiltration by three kinds of ultrafiltration membranes, obtains four sections
The polypeptide mixed liquor of different molecular weight, not only avoids the waste of active peptides in zymotic fluid, can also more refine, area
Not Hua ground study the biological activity of segment polypeptide between each molecular weight area；The essential amino acid composition of sea cucumber polypeptide be glycine and
Alanine, sea cucumber polypeptide is to DPPH, OH and O2-With preferable elimination effect, while sea cucumber polypeptide can significantly reduce blood
The concentration of total cholesterol, triglycerides in cleer and peaceful liver, can also promote NIH/3T3 cell growths, to NIH/3T3 cell collagens
The secretion of albumen has significant facilitation, also with certain anti-oxidative damage ability, the work(for reducing blood glucose, improving sugar tolerance
Effect.
Fermentation synergist in pre-treatment step includes the substance of following content：72% urea, 6% α-naphthaleneacidsodium, 18% oxygen
Change calcium, 3% magnesium sulfate, 1% dimethyl phosphate；The α-naphthaleneacidsodium Yu dimethyl phosphate of special proportioning have association in fermentation synergist
Same-action, which can promote S. cervisiae to enhance the secretion to acetyl coenzyme A, and then promote itself and oxaloacetic acid
Condensation reaction, accelerate the synthesis of citric acid, can increase substantially the speed of S. cervisiae tricarboxylic acid cycle, accelerate wine brewing
The growth and breeding and fermentating metabolism of saccharomycete promote S. cervisiae to be metabolized out more enzymes and are acted on fermentation substrate, Jin Erti
The yield and yield of high sea cucumber polypeptide.
Embodiment 4：
DPPH free radical scavenging activities measure：
Suitable concentration sea cucumber polypeptide liquid is taken respectively（Selected from embodiment 3）1mL measures T1 in test tube（>10kDa）、T2（10~
3kDa）、T3（3~1kDa）With T4（<1kDa）The clearance rate of four molecule sections, peptide concentration 1mg/mL, 5mg/mL, 15mg/
ML, 25mg/mL, 50mg/mL, 75mg/mL are purged rate measurement.
Compound concentration is 1 × 10-495% ethanol solutions of mol/L DPPH are added in test tube and are uniformly mixed, at dark often
Temperature is protected from light 30min, and control group is separately taken to replace sample, blank group to replace DPPH with equivalent ethanol solution with equivalent distilled water
Solution is used in combination VC as positive control, absorbance is measured in the case where wavelength is the ultraviolet specrophotometer of 517nm, by following public affairs
Formula calculates free radical scavenging activity（D）：
,
Wherein it is for AXSample-adding group absorbance, AYFor blank group absorbance, ACFor control group absorbance, it is clear to calculate DPPH free radicals
Except rate measurement result is as shown in table 1, as shown in Table 1, sea cucumber polypeptide has stronger scavenging effect, Er Qieqi to DPPH free radicals
Clearance rate has dose dependent, also knows T3（3-1kDa）The DPPH free radical scavenging activity effects of the sea cucumber polypeptide of molecule section are strong
With other molecule section.
Table 1.DPPH free radical scavenging activity results
Embodiment 5：
Scavenging action to hydroxyl free radical measures：
Suitable concentration sea cucumber polypeptide liquid is taken respectively（Selected from embodiment 3）1mL measures T1 in test tube（>10kDa）、T2（10~
3kDa）、T3（3~1kDa）With T4（<1kDa）The clearance rate of four molecule sections, peptide concentration 1mg/mL, 3mg/mL, 5mg/
ML, 10mg/mL, 15mg/mL are purged rate measurement.
Clean test tube with ground stopper is taken to sequentially add 1.0mL Phen aqueous solutions（7.5mmol/L）、5.0mLPBS（pH7.4）、
1.0mLFeSO4Solution（7.5mmol/L）, 1mL0.1%H2O2 solution and 1mL different molecular segment polypeptide solution, place at room temperature
60min surveys absorbance at wavelength 510nm, is denoted as chemical feed pipe As, using VC as positive control, 1mLH is replaced with 1mL distilled water2O2
Its absorbance is surveyed, is denoted as and does not damage pipe Ab；1mL sea cucumber polypeptides are replaced to survey its absorbance with the distilled water of 1mL, note damages pipe Ap,
Sea cucumber polypeptide is calculated by formula respectively in T1（>10kDa）、T2（10~3kDa）、T3（3~1kDa）With T4（<1kDa）Four points
The size of the Scavenging action to hydroxyl free radical D of subsegment：
,
If sample concentration is sets sample concentration as 1mg/mL, 3mg/mL, 5mg/mL, 10mg/mL, 15mg/mL, research is dense with sample
Degree increases, and the variation of Scavenging action to hydroxyl free radical, Scavenging action to hydroxyl free radical measurement result is as shown in table 2, as shown in Table 2, in institute
In the sea cucumber polypeptide for having four segment molecule sections, T3（3~1kDa）To the elimination efficiency highest of hydroxy radical, while being also seen that sea
Join polypeptide has dose dependent to the scavenging effect of hydroxy radical.
2. Scavenging action to hydroxyl free radical result of table
Routine operation in operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of sea cucumber polypeptide, it is characterised in that：The preparation method of the sea cucumber polypeptide includes：
Pretreatment：First sea cucumber is cleaned with clear water, then is dipped to sea cucumber with clear water and eases back, flowing water dries after rinsing, by solid-liquid ratio
1:10~12 homogenate, low temperature drying to water content are 35~45%, and fermentation synergist is added according to sea cucumber dry weight 0.2~0.4%, mixes
It closes uniformly up to fermentation substrate；
Fermentation：Aspergillus oryzae seed liquor, S. cervisiae seed liquor is taken to be inoculated in fermentation substrate and ferment；It boils and goes out after fermentation
Enzyme, centrifuging and taking supernatant cross miillpore filter, and filtrate is zymotic fluid；
Enzymolysis：It is 75~80% by zymotic fluid low temperature drying to water content, addition neutral proteinase, (R) -1,1 '-dinaphthalene -2,2 ' -
Phenolic group diisopropyl oxygen titanium digests, and boiling enzyme deactivation after enzymolysis obtains enzymolysis liquid；
Ultrafiltration：Clear enzymolysis liquid is obtained after taking enzymolysis liquid to clean, with the ultrafiltration membrane that molecular cut off is 10KDa, 3KDa, 1kDa
Classified filtering zymotic fluid obtains the polypeptide mixed liquor component of four sections of different molecular weights：T1 is>10kDa, T2 are 10~3kDa, T3
It is for 3~1kDa, T4<1kDa；It is freeze-dried after concentration up to sea cucumber polypeptide sterling.
2. a kind of sea cucumber polypeptide according to claim 1, it is characterised in that：In the pre-treatment step, fermentation synergist
Include the substance of following content：65~80% urea, 2~6% α-naphthaleneacidsodiums, 15~20% calcium oxide, 3~7% magnesium sulfate, 1~
5% dimethyl phosphate, total amount 100%.
3. a kind of sea cucumber polypeptide according to claim 1, it is characterised in that：In the fermentation step, aspergillus oryzae seed liquor
Additive amount be the 4~5% of fermentation substrate quality, the additive amount of S. cervisiae seed liquor is the 2~4% of fermentation substrate quality.
4. a kind of sea cucumber polypeptide according to claim 1, it is characterised in that：In the fermentation step, shaking speed of fermenting
For 140~160r/min, fermentation temperature is 28~30 DEG C, and fermentation time is 48~72h.
5. a kind of sea cucumber polypeptide according to claim 1, it is characterised in that：In the fermentation step, the hole of miillpore filter
Diameter is 0.2~0.25 μm.
6. a kind of sea cucumber polypeptide according to claim 1, it is characterised in that：In the enzymolysis step, neutral proteinase
Additive amount is 3000~4500U/g, and the additive amount of (R) -1,1 '-dinaphthalene -2,2 '-phenolic group diisopropyl oxygen titaniums are neutral proteinase weight
1.5~the 2.3 ‰ of amount.
7. a kind of sea cucumber polypeptide according to claim 1, it is characterised in that：In the enzymolysis step, hydrolysis temperature 55
~58 DEG C, enzymolysis pH value is 7.2~7.4, and enzymolysis time is 1.5~2.0h.
8. a kind of sea cucumber polypeptide according to claim 1, it is characterised in that：In the ultrafiltration step, enzymolysis liquid removal of impurities behaviour
As：Activated carbon, macroporous resin adsorption grease, impurity particle and desalination are sequentially added in enzymolysis liquid, after removed with sock filtration
Adsorbent is removed, filter is added and is removed by filtration activated carbon and excess oil.
9. a kind of sea cucumber polypeptide according to claim 1, it is characterised in that：In the ultrafiltration step, the temperature of freeze-drying
Degree is -10~-20 DEG C, and vacuum degree is 50~100Pa, and sublimation drying is 48~60h.
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| CN201810292047.8A CN108660177A (en) | 2018-04-03 | 2018-04-03 | A kind of sea cucumber polypeptide |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110184320A (en) * | 2019-06-19 | 2019-08-30 | 青岛代山慧海生态科技有限公司 | A kind of preparation method of compound sea cucumber polypeptide |
| CN113265437A (en) * | 2021-06-01 | 2021-08-17 | 大连工业大学 | Preparation method and application of sea cucumber protein peptide for promoting proliferation and differentiation of MC3T3-E1 cells |
-
2018
- 2018-04-03 CN CN201810292047.8A patent/CN108660177A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110184320A (en) * | 2019-06-19 | 2019-08-30 | 青岛代山慧海生态科技有限公司 | A kind of preparation method of compound sea cucumber polypeptide |
| CN113265437A (en) * | 2021-06-01 | 2021-08-17 | 大连工业大学 | Preparation method and application of sea cucumber protein peptide for promoting proliferation and differentiation of MC3T3-E1 cells |
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