CN107385000A - The method of abstraction function small peptide from deep-sea fish - Google Patents
The method of abstraction function small peptide from deep-sea fish Download PDFInfo
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- CN107385000A CN107385000A CN201710643602.2A CN201710643602A CN107385000A CN 107385000 A CN107385000 A CN 107385000A CN 201710643602 A CN201710643602 A CN 201710643602A CN 107385000 A CN107385000 A CN 107385000A
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- small peptide
- deep
- enzymolysis
- function small
- sea fish
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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
- 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
- C07K1/30—Extraction; Separation; Purification by precipitation
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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
-
- 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
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
Abstract
The invention discloses the method for the abstraction function small peptide from deep-sea fish, including stepwise discretization, isoelectric precipitation, second ultrafiltration and nanofiltration and freeze-drying.Digested using marine protein enzyme and land proteinase synergy, first using Porphyra yezoensis protease, continue to digest using flavor protease on this basis, can obtain the function small peptide that collagen can be protected not decomposed by hydrochloric acid in gastric juice.Have the beneficial effect that:Connected between obtained function small peptide and collagen by intermolecular force and hydrogen bond; function small peptide is in collagen molecules surface attachment; wrapped up, collagen can be protected not decomposed by hydrochloric acid in gastric juice, and it can be promoted to reach animal connective tissue, cartilage and dermal layer of the skin.
Description
Technical field
The present invention relates to small peptide preparation field, the method for specifically a kind of abstraction function small peptide from deep-sea fish.
Background technology
Naturally occurring Marine Bioactive Peptides due in organism content it is low, and extract difficult, it is difficult to realize a large amount of
Needed for production supply, chemical artificial synthetic polypeptide is costly.So sight is more invested exploitation proteolysis production by people
Come in this approach of thing acquisition active peptides.Because the environment and terrestrial life of marine organisms existence are entirely different, such as high pressure, low
The extreme environment such as temperature, high temperature and high salt, in order to adapt to these extreme marine biological environments, marine protein matter no matter amino
The composition or the sequence of amino acid of acid are all very different with terrestrial life albumen, meanwhile, marine protein resource without
By being all far longer than land protein resource in type and quantity, and do not developed well.Miscellaneous ocean egg
In casamino acid sequence, potential many amino acid sequences with bioactivity, with special protease hydrolytic, just discharge
Active peptide fragment.Marine protein resource is the important of the important albumen group food of the 21 century mankind and bioactive substance
Source.The current marine protein total resources in China comes out at the top in countries in the world, but the protide aquatic products that China is current
In product, in addition to a part is directly edible, major part is made food by simple process technology and enters market, and process technology falls
Afterwards, product structure is single, and value-added content of product is low so that the competitiveness of product in the international market is poor.Therefore, China is badly in need of
Marine protein resource is optimized and utilized and high-valued processing.
Collagen is also known as collagen, is the fibrous proteins being mutually wound by three peptide chains, is a kind of glycoprotein,
Containing substantial amounts of glycine, proline, hydroxyproline and sugar, the tissue such as skin, bone, cartilage and tendon of animal is widely present in
In, it is the most class protein of body content.Collagen can be used for repairing the sides such as skin scar, anti-ageing, light spot and whitening
Face, it is beauty sage's product, but by edible way replenishing collagen, its utilization rate is extremely low, can largely be decomposed by body, loses
Deactivation.
The content of the invention
It is an object of the invention to provide a kind of method of the abstraction function small peptide from deep-sea fish, the function of extracting to obtain is small
Peptide can protect collagen not decomposed by hydrochloric acid in gastric juice, and it can be promoted to reach animal connective tissue, cartilage and dermal layer of the skin.
The present invention is directed to the problem of being mentioned in background technology, and the technical scheme taken is:Abstraction function is small from deep-sea fish
The method of peptide, including stepwise discretization, isoelectric precipitation, second ultrafiltration and nanofiltration and freeze-drying.Stepwise discretization step is in depth
Pure water homogenate is added in ocean fish minced fillet, adds Porphyra yezoensis protease and bioactive peptide enzymolysis, flavor egg is added after adjusting pH
White enzyme enzymolysis, obtains enzymolysis liquid.The composition of marine protein matter amino acid or the sequence of amino acid are all given birth to land
Thing albumen is very different, and many different active peptide segments are can release with marine protein enzyme enzymolysis marine product.First use bar
Spot seaweed protease, continue to digest using flavor protease on this basis, can obtain can protect collagen not by hydrochloric acid in gastric juice point
The function small peptide of solution.Do not have at present and obtain the report of above-mentioned function small peptide using other protease hydrolyzeds.Using marine protein enzyme
Digest, have significant creative with land proteinase synergy.
20 ~ 30 times of volume of water are added in deep-sea fish minced fillet, the addition of Porphyra yezoensis protease is 5 ~ 8wt ‰, enzymolysis temperature
25 ~ 35 DEG C of degree, pH are 7.0 ~ 9.0, and enzymolysis time is 30 ~ 40min, and enzymolysis terminates rear enzyme deactivation.Under above-mentioned enzymatic hydrolysis condition, streak is purple
The enzymolysis efficiency of dish protease is high will not to cause the wasting of resources again, for most preferably condition.
Flavor protease addition is 1 ~ 3wt ﹪, and enzymolysis pH is 6.0 ~ 7.0, and hydrolysis temperature is 52 ~ 60 DEG C, enzymolysis time
For 15 ~ 25min, enzymolysis terminates rear enzyme deactivation, filters to take filtrate.Under above-mentioned enzymatic hydrolysis condition, the enzymolysis efficiency height of flavor protease is again
The wasting of resources will not be caused, for most preferably condition.
The addition of bioactive peptide is 10 ~ 20 ﹪ of Porphyra yezoensis protease quality, and amino acid sequence is
HSHACTSYYHPCSKLLCVCANCSR.Above-mentioned bioactive peptide can change Porphyra yezoensis egg by Van der Waals force and electrostatic interaction
The tertiary structure of white enzyme, makes the enzymolysis vigor of Porphyra yezoensis protease be improved significantly.
For isoelectric precipitation step for enzymolysis liquid is filtered, the pH value for adjusting filtrate is 4.5 ~ 4.8, and centrifuging and taking is sunk after standing
Form sediment.Under above-mentioned pH environment, function small peptide is in electroneutral, is slightly soluble in water, most of to be isolated with precipitation form from the aqueous solution
Come.
Second ultrafiltration and nano-filtration step are first to use 30 ~ 40kD of retention relative molecular mass film ultrafiltration, then using retention
4 ~ 8kD of relative molecular mass film ultrafiltration, filtered solution is finally subjected to nanofiltration desalination.In order to prevent Pore Blocking or membrane damage,
Ultrafiltration is first carried out using big porous ultrafiltration membrane, then using small porous ultrafiltration membrane ultrafiltration, effectively removes the impurity that molecular weight is more than 4 ~ 8kD.
Nanofiltration can be more thoroughly remove solution in inorganic salts and molecular weight be less than 200Da impurity, function small peptide is further carried out
Purifying.
It is freeze-dried in step and adds 20 ~ 35wt% polyethylene Topiramate Los oxazolidinone as lyophilisation additives, is freeze-dried and obtains work(
Can small peptide freeze-dried powder.Function small peptide need containing suitable residual moisture come be maintained at storage during property stabilization, excessively
Drying by the hydrogen bond for making protein molecule surface and polar group exposure and be denatured.In freezing dry process, polyethylene is added
Topiramate Los oxazolidinone, can the albumen stretching, extension of defencive function small peptide, quaternary structure, so as to being protected to the small peptide activity of function.Above-mentioned work(
It can be connected between small peptide and collagen by intermolecular force and hydrogen bond, function small peptide is attached on collagen molecules surface
, wrapped up, collagen can be protected not decomposed by hydrochloric acid in gastric juice, and it can be promoted to reach animal connective tissue, cartilage and skin
Skin corium, the specific mechanism of action are still not clear.
Compared with prior art, the advantage of the invention is that:
1. the composition of marine protein matter amino acid or the sequence of amino acid all have very big with terrestrial life albumen
Difference, many different active peptide segments are can release with marine protein enzyme enzymolysis marine product.Porphyra yezoensis protease is first used,
Continue using dividing flavor protease to digest, to can obtain the function small peptide that collagen can be protected not decomposed by hydrochloric acid in gastric juice on the basis of this.
Do not have at present and obtain the report of above-mentioned function small peptide using other protease hydrolyzeds.Assisted using marine protein enzyme and land protease
With enzymolysis, have significant creative.
2. amino acid sequence, which is HSHACTSYYHPCSKLLCVCANCSR bioactive peptide, can pass through Van der Waals force and electrostatic
Act on to change the tertiary structure of Porphyra yezoensis protease, the enzymolysis vigor of Porphyra yezoensis protease is improved significantly.
3. being connected between above-mentioned function small peptide and collagen by intermolecular force and hydrogen bond, function small peptide is in glue
Former protein molecular surface attachment, is wrapped up, and collagen can be protected not decomposed by hydrochloric acid in gastric juice, and it can be promoted to reach animal knot
Tissue, cartilage and dermal layer of the skin are formed, the specific mechanism of action is still not clear.
Embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
The method of abstraction function small peptide from deep-sea fish, including stepwise discretization, isoelectric precipitation, second ultrafiltration and nanofiltration and freezing
Dry.Stepwise discretization step is that pure water homogenate is added in deep-sea fish minced fillet, adds Porphyra yezoensis protease and bioactive peptide
Enzymolysis, flavor protease enzymolysis is added after adjusting pH, obtains enzymolysis liquid.Marine protein matter no matter amino acid composition or
The sequence of amino acid is all very different with terrestrial life albumen, and many is can release not with marine protein enzyme enzymolysis marine product
Same active peptide segment.Porphyra yezoensis protease is first used, continues to digest using flavor protease on this basis, can obtain to protect
Protect the function small peptide that collagen is not decomposed by hydrochloric acid in gastric juice.Do not have at present and obtain above-mentioned function small peptide using other protease hydrolyzeds
Report.Digested, had significant creative using marine protein enzyme and land proteinase synergy.
20 ~ 30 times of volume of water are added in deep-sea fish minced fillet, the addition of Porphyra yezoensis protease is 5 ~ 8wt ‰, enzymolysis temperature
25 ~ 35 DEG C of degree, pH are 7.0 ~ 9.0, and enzymolysis time is 30 ~ 40min, and enzymolysis terminates rear enzyme deactivation.Under above-mentioned enzymatic hydrolysis condition, streak is purple
The enzymolysis efficiency of dish protease is high will not to cause the wasting of resources again, for most preferably condition.
Flavor protease addition is 1 ~ 3wt ﹪, and enzymolysis pH is 6.0 ~ 7.0, and hydrolysis temperature is 52 ~ 60 DEG C, enzymolysis time
For 15 ~ 25min, enzymolysis terminates rear enzyme deactivation, filters to take filtrate.Under above-mentioned enzymatic hydrolysis condition, the enzymolysis efficiency height of flavor protease is again
The wasting of resources will not be caused, for most preferably condition.
The addition of bioactive peptide is 10 ~ 20 ﹪ of Porphyra yezoensis protease quality, and amino acid sequence is
HSHACTSYYHPCSKLLCVCANCSR.Above-mentioned bioactive peptide can change Porphyra yezoensis egg by Van der Waals force and electrostatic interaction
The tertiary structure of white enzyme, makes the enzymolysis vigor of Porphyra yezoensis protease be improved significantly.
For isoelectric precipitation step for enzymolysis liquid is filtered, the pH value for adjusting filtrate is 4.5 ~ 4.8, and centrifuging and taking is sunk after standing
Form sediment.Under above-mentioned pH environment, function small peptide is in electroneutral, is slightly soluble in water, most of to be isolated with precipitation form from the aqueous solution
Come.
Second ultrafiltration and nano-filtration step are first to use 30 ~ 40kD of retention relative molecular mass film ultrafiltration, then using retention
4 ~ 8kD of relative molecular mass film ultrafiltration, filtered solution is finally subjected to nanofiltration desalination.In order to prevent Pore Blocking or membrane damage,
Ultrafiltration is first carried out using big porous ultrafiltration membrane, then using small porous ultrafiltration membrane ultrafiltration, effectively removes the impurity that molecular weight is more than 4 ~ 8kD.
Nanofiltration can be more thoroughly remove solution in inorganic salts and molecular weight be less than 200Da impurity, function small peptide is further carried out
Purifying.
It is freeze-dried in step and adds 20 ~ 35wt% polyethylene Topiramate Los oxazolidinone as lyophilisation additives, is freeze-dried and obtains work(
Can small peptide freeze-dried powder.Function small peptide need containing suitable residual moisture come be maintained at storage during property stabilization, excessively
Drying by the hydrogen bond for making protein molecule surface and polar group exposure and be denatured.In freezing dry process, polyethylene is added
Topiramate Los oxazolidinone, can the albumen stretching, extension of defencive function small peptide, quaternary structure, so as to being protected to the small peptide activity of function.Above-mentioned work(
It can be connected between small peptide and collagen by intermolecular force and hydrogen bond, function small peptide is attached on collagen molecules surface
, wrapped up, collagen can be protected not decomposed by hydrochloric acid in gastric juice, and it can be promoted to reach animal connective tissue, cartilage and skin
Skin corium, the specific mechanism of action are still not clear.
Embodiment 2:
The most preferred method of abstraction function small peptide, comprises the following steps from deep-sea fish:
1)Stepwise discretization:Pure water homogenate is added in deep-sea fish minced fillet, adds Porphyra yezoensis protease and bioactive peptide enzymolysis,
Flavor protease enzymolysis is added after adjusting pH, obtains enzymolysis liquid.26 times of volume of water, Porphyra yezoensis albumen are added in deep-sea fish minced fillet
The addition of enzyme is 6wt ‰, 32 DEG C, pH 8.0, enzymolysis time 34min of hydrolysis temperature, digests and terminates rear enzyme deactivation.Flavor egg
White enzyme addition is 2wt ﹪, and enzymolysis pH is 6.4, and hydrolysis temperature is 55 DEG C, enzymolysis time 21min, and enzymolysis terminates rear enzyme deactivation,
Filter to take filtrate.The addition of bioactive peptide is 16 ﹪ of Porphyra yezoensis protease quality, and amino acid sequence is
HSHACTSYYHPCSKLLCVCANCSR;
2)Isoelectric precipitation:Enzymolysis liquid is filtered, the pH value for adjusting filtrate is 4.7, and centrifuging and taking precipitates after standing;
3)Second ultrafiltration and nanofiltration:First using retention relative molecular mass 35kD film ultrafiltration, then using retention average molecular matter
6kD film ultrafiltration is measured, filtered solution is finally subjected to nanofiltration desalination;
4)Freeze-drying:30wt% polyethylene Topiramate Los oxazolidinone is added in filtered solution after nanofiltration desalination as lyophilisation additives, freezing
It is dried to obtain function small peptide freeze-dried powder.Function small peptide needs to be maintained at property during storage containing suitable residual moisture
Stable, the hydrogen bond for making protein molecule surface and polar group exposure are denatured by excessive drying.In freezing dry process, add
Enter polyethylene Topiramate Los oxazolidinone, can the albumen stretching, extension of defencive function small peptide, quaternary structure, so as to being protected to the small peptide activity of function
Shield.Connected between above-mentioned function small peptide and collagen by intermolecular force and hydrogen bond, function small peptide is in collagen point
Sub- surface attachment, is wrapped up, and collagen can be protected not decomposed by hydrochloric acid in gastric juice, and it can be promoted to reach animal connective tissue, soft
Bone and dermal layer of the skin, the specific mechanism of action are still not clear.
Embodiment 3:
The most preferred method of abstraction function small peptide, comprises the following steps from deep-sea fish:
1)Stepwise discretization:Pure water homogenate is added in deep-sea fish minced fillet, adds Porphyra yezoensis protease and bioactive peptide enzymolysis,
Flavor protease enzymolysis is added after adjusting pH, obtains enzymolysis liquid.30 times of volume of water, Porphyra yezoensis albumen are added in deep-sea fish minced fillet
The addition of enzyme is 5.5wt ‰, 32 DEG C, pH 8.0, enzymolysis time 34min of hydrolysis temperature, digests and terminates rear enzyme deactivation.Flavor
Protease addition is 1.8wt ﹪, and enzymolysis pH is 6.4, and hydrolysis temperature is 55 DEG C, enzymolysis time 21min, and enzymolysis goes out after terminating
Enzyme, filter to take filtrate.The addition of bioactive peptide is 16 ﹪ of Porphyra yezoensis protease quality, and amino acid sequence is
HSHACTSYYHPCSKLLCVCANCSR;
2)Isoelectric precipitation:Enzymolysis liquid is filtered, the pH value for adjusting filtrate is 4.7, and centrifuging and taking precipitates after standing;
3)Second ultrafiltration and nanofiltration:First using retention relative molecular mass 35kD film ultrafiltration, then using retention average molecular matter
6kD film ultrafiltration is measured, filtered solution is finally subjected to nanofiltration desalination;
4)Freeze-drying:30wt% polyethylene Topiramate Los oxazolidinone is added in filtered solution after nanofiltration desalination as lyophilisation additives, freezing
It is dried to obtain function small peptide freeze-dried powder.Function small peptide needs to be maintained at property during storage containing suitable residual moisture
Stable, the hydrogen bond for making protein molecule surface and polar group exposure are denatured by excessive drying.In freezing dry process, add
Enter polyethylene Topiramate Los oxazolidinone, can the albumen stretching, extension of defencive function small peptide, quaternary structure, so as to being protected to the small peptide activity of function
Shield.Connected between above-mentioned function small peptide and collagen by intermolecular force and hydrogen bond, function small peptide is in collagen point
Sub- surface attachment, is wrapped up, and collagen can be protected not decomposed by hydrochloric acid in gastric juice, and it can be promoted to reach animal connective tissue, soft
Bone and dermal layer of the skin, the specific mechanism of action are still not clear.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme is described in detail embodiment described above, it should be understood that it is described above only
For the specific embodiment of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should be included in the scope of the protection.
SEQUENCE LISTING
<110>The Gothic Bioisystech Co., Ltd of Lanxi City
<120>The method of abstraction function small peptide from deep-sea fish
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> PRT
<213>It is artificial synthesized
<400> 1
His Ser His Ala Cys Thr Ser Tyr Tyr His Pro Cys Ser Lys Leu Leu
1 5 10 15
Cys Val Cys Ala Asn Cys Ser Arg
20
Claims (7)
1. the method for abstraction function small peptide from deep-sea fish, including stepwise discretization, isoelectric precipitation, second ultrafiltration and nanofiltration and cold
It is lyophilized dry, it is characterised in that:Described stepwise discretization step is that pure water homogenate is added in deep-sea fish minced fillet, adds streak purple
Dish protease and bioactive peptide enzymolysis, flavor protease enzymolysis is added after adjusting pH, obtains enzymolysis liquid.
2. the method for the abstraction function small peptide according to claim 1 from deep-sea fish, it is characterised in that:Described deep-sea fish
Add 20 ~ 30 times of volume of water in minced fillet, the addition of Porphyra yezoensis protease is 5 ~ 8wt ‰, 25 ~ 35 DEG C of hydrolysis temperature, and pH is
7.0 ~ 9.0, enzymolysis time is 30 ~ 40min, and enzymolysis terminates rear enzyme deactivation.
3. the method for the abstraction function small peptide according to claim 1 from deep-sea fish, it is characterised in that:Described flavor egg
White enzyme addition is 1 ~ 3wt ﹪, and enzymolysis pH is 6.0 ~ 7.0, and hydrolysis temperature is 52 ~ 60 DEG C, and enzymolysis time is 15 ~ 25min, enzymolysis
Enzyme deactivation after end, filters to take filtrate.
4. the method for the abstraction function small peptide according to claim 1 from deep-sea fish, it is characterised in that:Described is active short
The addition of peptide is 10 ~ 20 ﹪, amino acid sequence HSHACTSYYHPCSKLLCVCANCSR of Porphyra yezoensis protease quality.
5. the method for the abstraction function small peptide according to claim 1 from deep-sea fish, it is characterised in that:Described isoelectric point
For settling step for enzymolysis liquid is filtered, the pH value for adjusting filtrate is 4.5 ~ 4.8, and centrifuging and taking precipitates after standing.
6. the method for the abstraction function small peptide according to claim 1 from deep-sea fish, it is characterised in that:Described is secondary super
Filter and nano-filtration step is first using retention relative molecular mass 30 ~ 40kD film ultrafiltration, then using retention relative molecular mass 4 ~
8kD film ultrafiltration, filtered solution is finally subjected to nanofiltration desalination.
7. the method for the abstraction function small peptide according to claim 1 from deep-sea fish, it is characterised in that:Described freezing is done
20 ~ 35wt% polyethylene Topiramate Los oxazolidinones are added in dry step as lyophilisation additives.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102154422A (en) * | 2010-12-22 | 2011-08-17 | 广西大学 | Method for extracting functional small peptides from low-value sea fish |
CN106319014A (en) * | 2016-10-14 | 2017-01-11 | 浙江双屿生物股份有限公司 | Extracting method for micro-molecule deep-sea fish polypeptides |
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2017
- 2017-07-31 CN CN201710643602.2A patent/CN107385000A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102154422A (en) * | 2010-12-22 | 2011-08-17 | 广西大学 | Method for extracting functional small peptides from low-value sea fish |
CN106319014A (en) * | 2016-10-14 | 2017-01-11 | 浙江双屿生物股份有限公司 | Extracting method for micro-molecule deep-sea fish polypeptides |
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Application publication date: 20171124 |