CN107604029A - The method that active peptides are prepared using the deep-sea fish flesh of fish - Google Patents
The method that active peptides are prepared using the deep-sea fish flesh of fish Download PDFInfo
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Abstract
The invention discloses a kind of method that active peptides are prepared using the deep-sea fish flesh of fish, including degreasing, first time deodorant, enzymolysis, second of deodorant, ultrafiltration, simultaneously desalination and freeze-drying of decolourizing.Using 1 ethyl 3（3 methylaminos）Propyl group carbodiimide activation chitosan, and be coupled to form lipase immobilization carrier with glutaraldehyde, recycle bioactive peptide to improve the physics and chemisorbed power between carrier and lipase, improve stability, enzymatic activity, heat resistance and the access times of lipase；Hydrolysis degree, enzyme digestion reaction speed and the type for changing end group of deep-sea fish minced fillet albumen can be improved using the complementation and synergy of thermolysin and chymotrypsin hydrolytic sites, hydrolysis has the active peptides of antitumor activity.
Description
Technical field
It is specifically a kind of to prepare active peptides using the deep-sea fish flesh of fish the present invention relates to bioactive substance preparation field
Method.
Background technology
Although localized cancer can successfully use surgical operation and radiation therapy treatment at present, chemotherapy is still normal
Rule treatment late period or the first choice of metastatic tumo(u)r.Lacked because chemotherapeutics has low selectivity, erious adverse reaction, multidrug resistance etc.
Fall into, largely limit its use.
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.Marine protein resource is the important albumen of the 21 century mankind
The important sources of group food and bioactive substance.Before the current marine protein total resources in China ranks in countries in the world
Thatch, but in the current protide aquatic products in China, in addition to a part is directly edible, be largely made up of simple process technology
Food enters market, process technology falls behind, and product structure is single, and value-added content of product is low so that product is in the international market
Competitiveness is poor.Therefore, China is badly in need of optimizing marine protein resource and utilized and high-valued processing.
The content of the invention
It is an object of the invention to provide a kind of method that active peptides are prepared using the deep-sea fish flesh of fish, the work being prepared
Property polypeptide it is white, no fishy smell, lipid content is very low, and molecular weight is non-specific in reinforcement in 1000 ~ 4000 dalton
Antitumor T lymphocyte specific immune response while immune be present.
The present invention is directed to the problem of being mentioned in background technology, and the technical scheme taken is：Prepared and lived using the deep-sea fish flesh of fish
Property polypeptide method, including degreasing, first time deodorant, enzymolysis, second of deodorant, ultrafiltration, decolourize and desalination and freeze-drying.
To active peptides compared with small-molecule chemical medicine, there is higher affinity and stronger specificity to target tumor, and
And adverse reaction is low, susceptibility of the tumour to other treatment method can also be increased；Compared with antibody, because their volumes are micro-
It is small, therefore be easier to penetrate into tissue, there is good prospect as antitumoral compounds.Wherein, second of deodorant step
To be inoculated with the saccharomyces cerevisiae of activation and Bifidobacterium in enzymolysis liquid, 1.5 ~ 3.5h of ferment at constant temperature at 32 ~ 43 DEG C.Saccharomyces cerevisiae
Addition with Bifidobacterium is respectively 0.5 ~ 1.0 ‰ and 0.8 ~ 1.8 ‰, buy respectively in Angel Yeast Co., Ltd and
Shandong sunflower bio tech ltd.Obvious synergy during the fermentation be present in saccharomyces cerevisiae and Bifidobacterium,
The fishy smell materials such as dimethyl methylamine, the oxosilane of ten dimethyl six and the oxosilane of ten dimethyl five are divided as nitrogen source or nitrogen source
Solution, the fishy smell of enzymolysis liquid is greatly reduced.Meanwhile tunning ethanol also has the function that deodorant, fishy smell can be reduced.
Defatting step is：The deep-sea fish flesh of fish is cut into small pieces, and adds 2 ~ 4 ‰ chitosan-immobilized lipase, adds 8 ~ 15 times
The distilled water of volume, constant temperature digests 0.8 ~ 2.5h at 25 ~ 50 DEG C, and enzymolysis terminates rear enzyme deactivation, is filtrated to get degreasing deep-sea fish fish
Meat.Fat in the deep-sea fish flesh of fish can be progressively hydrolyzed into glycerine and aliphatic acid by lipase.Lipase after immobilization, it is stable
Property, enzymatic activity, heat resistance are significantly improved, and reusable.
The preparation process of chitosan-immobilized lipase is：With drying Chitosan powder, with the 60 of pH6.0 ~ 6.8 ~
80mmol/L phosphate buffer soaked overnights, then add the 1- ethyls -3- of Chitosan powder quality 0.7 ~ 1.2%（3- first
Base amino）Propyl group 0.5 ~ 0.9h of carbodiimide activation, add the glutaraldehyde coupling reaction 1 of 1 ~ 2 times of volume of Chitosan powder ~
1.8h, cleaned 5 ~ 8 times with distilled water, 0.7 ~ 2.0mg/mL lipase solutions and 0.1 ~ 0.3 ‰ bioactive peptides are added, in ice bath
0.5 ~ 1.5h is slowly stirred, then places refrigerator overnight, secondary daily distilled water cleaning down, filtration drying obtains chitosan
Immobilized lipase.1- ethyls -3-（3- methylaminos）Propyl group carbodiimide carries out chemical modification, effective activation to chitosan
Chitosan surface hydroxyl groups.Chitosan surface amino groups after activation are coupled to obtain excellent lipase immobilization load with glutaraldehyde
Body.Lipase adsorption forms chitosan-immobilized lipase on carrier.
The amino acid sequence of bioactive peptide is SCASVCKARCLRARGCRCVSVYCRCLR.Above-mentioned bioactive peptide can be obvious
Strengthen the physics and chemisorption power between lipase and lipase immobilization carrier, improve immobilized lipase enzymatic conversion
Rate, and improve the reusable number of chitosan-immobilized lipase.
First time deodorant step is：The degreasing deep-sea fish flesh of fish is dipped in 0.4 ~ 0.8h in deodorant liquid, soaking temperature is 0 ~ 4
DEG C, deodorant liquid composition and its parts by weight are 0.2 ~ 0.8 part of 3 ~ 8 parts of Tea Polyphenols, 10 ~ 20 parts of pure water and grape seed extract.It is above-mentioned
Composition acts synergistically in deodorant liquid, has various biological activity, can eliminate methyl mercaptan compound, and can be with amino acid
With reference to, there is certain passivation enzyme and bactericidal action, and composition is natural, it is green safe.
Enzymolysis step is：The flesh of fish after first time deodorant is beaten into minced fillet, adds the distilled water of 2 ~ 4 times of volumes, is added
0.1 ~ 0.3% thermolysin and 0.2 ~ 0.5% chymotrypsin, in 27 ~ 40 DEG C, under natural ph digest 1.5 ~
3.0h, 4 ~ 8 ‰ flavor proteases are added after enzyme deactivation, in 45 ~ 55 DEG C, 0.5 ~ 1.0h is digested under the conditions of pH6.0 ~ 6.8, filters to take filter
Liquid.Deep-sea fish minced fillet can be improved using the complementation and synergy of thermolysin and chymotrypsin hydrolytic sites
Hydrolysis degree, enzyme digestion reaction speed and the type for changing end group of albumen, hydrolysis have the active peptides of antitumor activity.Wind
Taste protease is exopeptidase, the peptide bond that the terminal amino acid of hydrolyzable peptide chain two is formed（Bitter peptides）, bitter peptide is thoroughly degraded
For amino acid.Meanwhile with the reduced sugar in food flavor enzyme Maillard reaction occurs for enzymolysis product and amino acid together, natural perfume is produced
Taste, therefore enzymolysis liquid has unique flavor and substantially reduces bitter taste caused by hydrolysis.
Ultrafiltration step retains 30 ~ 40kD of relative molecular mass film ultrafiltration first to use, then using retention average molecular matter
Measure 4 ~ 8kD film ultrafiltration.In order to prevent Pore Blocking or membrane damage, ultrafiltration is first carried out using big porous ultrafiltration membrane, then using aperture
Milipore filter ultrafiltration, effectively remove the impurity that molecular weight is more than 4 ~ 8kD.
Decolourize and desalting steps are：0.5 ~ 1.5% activated carbon, at 60 ~ 70 DEG C, 100 ~ 200r/ of rotating speed are added in ultrafiltrate
Decolourized 0.4 ~ 0.8h under the conditions of min, filters to take filtrate, and filtrate finally is carried out into nanofiltration desalination.Activated carbon has the poroid of densification
Structure, there is excellent adsorptivity to pigment and fishy smell material, make the active peptides freeze-dried powder that finally obtains white, no raw meat
Taste.Part below 500 dalton is made up of oligopeptides and amino acid, and the bioactivity of this constituent part is poor, and major part is
Bitter peptides.Nanofiltration can effectively remove the inorganic salts in impurity and solution below 500 dalton, progress further to active peptides
Purifying.
Step is freeze-dried to add 20 ~ 35wt% polyethylene Topiramate Los oxazolidinones in the enzymolysis liquid after nanofiltration desalination, freezing is done
It is dry to obtain active peptides freeze-dried powder.Active peptides combination antigenic action immune cell activated, promotes it in the immune system of body
Differentiation and maturation, the activity and quantity of immunocyte are improved, strengthen itself antitumor ability.Above-mentioned active peptides are in reinforcement
Antitumor T lymphocyte specific immune response while nospecific immunity be present.Active peptides can suppress HL-60 increasings
Simultaneously inducing cell apoptosis is grown, its mechanism may be relevant with promoting the up-regulation of p53 protein expressions, Bcl-2 expression downwards, but specific mechanism
It is and indefinite.
Compared with prior art, the advantage of the invention is that：
1. using 1- ethyls -3-（3- methylaminos）Propyl group carbodiimide activation chitosan, and be coupled to form fat with glutaraldehyde
Enzyme immobilization carrier, the bioactive peptide that amino acid sequence is SCASVCKARCLRARGCRCVSVYCRCLR is recycled to be carried to improve
Physics and chemisorbed power between body and lipase, improve stability, enzymatic activity, heat resistance and the access times of lipase；
2. deep-sea fish can be improved using the complementation and synergy of thermolysin and chymotrypsin hydrolytic sites
Hydrolysis degree, enzyme digestion reaction speed and the type for changing end group of minced fillet albumen, hydrolysis have the activity of antitumor activity more
Peptide.Flavor protease is exopeptidase, the peptide bond that the terminal amino acid of hydrolyzable peptide chain two is formed（Bitter peptides）, bitter peptide is thorough
It is degraded to amino acid.Meanwhile with the reduced sugar in food flavor enzyme Maillard reaction occurs for enzymolysis product and amino acid together, day is produced
Right fragrance, therefore enzymolysis liquid has unique flavor and substantially reduces bitter taste caused by hydrolysis；
3. obtained active peptides combination antigenic action immune cell activated, promotes it to be divided into the immune system of body
It is ripe, the activity and quantity of immunocyte are improved, strengthens itself antitumor ability.Active peptides non-specificity in reinforcement is exempted from
Antitumor T lymphocyte specific immune response while epidemic disease be present.Active peptides can suppress HL-60 and breed and induce thin
Born of the same parents' apoptosis, its mechanism may be relevant with promoting the up-regulation of p53 protein expressions, Bcl-2 expression downward, but specifically mechanism and indefinite；
4. first time is dipped in deodorant liquid deodorant, methyl mercaptan compound is eliminated；Second of saccharomyces cerevisiae and Bifidobacterium collaboration hair
Ferment deodorant, fishy smell material are decomposed as nitrogen source or nitrogen source, and the fishy smell of enzymolysis liquid is greatly reduced.Finally, using activated carbon decolorizing
While play deodorant effect, therefore, obtained active peptides are without fishy smell.
Embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
The method that active peptides are prepared using the deep-sea fish flesh of fish, is comprised the following steps：
1）Degreasing：The deep-sea fish flesh of fish is cut into small pieces, and adds 2 ~ 4 ‰ chitosan-immobilized lipase, adds the steaming of 8 ~ 15 times of volumes
Distilled water, constant temperature digests 0.8 ~ 2.5h at 25 ~ 50 DEG C, and enzymolysis terminates rear enzyme deactivation, is filtrated to get degreasing deep-sea fish flesh of fish degreasing.Shell
The preparation process of chitosan-immobilized lipase is：With Chitosan powder is dried, with 60 ~ 80mmol/L phosphate of pH6.0 ~ 6.8
Buffer solution soaked overnight, then add the 1- ethyls -3- of Chitosan powder quality 0.7 ~ 1.2%（3- methylaminos）Propyl group carbon two
Imines activates 0.5 ~ 0.9h, adds the glutaraldehyde 1 ~ 1.8h of coupling reaction of 1 ~ 2 times of volume of Chitosan powder, is cleaned with distilled water
5 ~ 8 times, 0.7 ~ 2.0mg/mL lipase solutions and 0.1 ~ 0.3 ‰ bioactive peptides are added, 0.5 ~ 1.5h is slowly stirred in ice bath
, refrigerator overnight, secondary daily distilled water cleaning down are then placed, filtration drying obtains chitosan-immobilized lipase.Activity
The amino acid sequence of small peptide is SCASVCKARCLRARGCRCVSVYCRCLR；
2）First time deodorant：The degreasing deep-sea fish flesh of fish is dipped in 0.4 ~ 0.8h in deodorant liquid, soaking temperature is 0 ~ 4 DEG C, deodorant liquid
Composition and its parts by weight are 0.2 ~ 0.8 part of 3 ~ 8 parts of Tea Polyphenols, 10 ~ 20 parts of pure water and grape seed extract.
3）Enzymolysis：The flesh of fish after first time deodorant is beaten into minced fillet, adds the distilled water of 2 ~ 4 times of volumes, add 0.1 ~
0.3% thermolysin and 0.2 ~ 0.5% chymotrypsin, in 27 ~ 40 DEG C, 1.5 ~ 3.0h is digested under natural ph, is gone out
4 ~ 8 ‰ flavor proteases are added after enzyme, in 45 ~ 55 DEG C, 0.5 ~ 1.0h is digested under the conditions of pH6.0 ~ 6.8, filters to take filtrate；
4）Second of deodorant step is that saccharomyces cerevisiae and the Bifidobacterium of activation, the constant temperature at 32 ~ 43 DEG C are inoculated with enzymolysis liquid
Ferment 1.5 ~ 3.5h.The addition of saccharomyces cerevisiae and Bifidobacterium is respectively 0.5 ~ 1.0 ‰ and 0.8 ~ 1.8 ‰；
5）Ultrafiltration：First using 30 ~ 40kD of retention relative molecular mass film ultrafiltration, then using 4 ~ 8kD of retention relative molecular mass
Film ultrafiltration；
6）Decolouring and desalination：0.5 ~ 1.5% activated carbon, at 60 ~ 70 DEG C, rotating speed 100 ~ 200r/min conditions are added in ultrafiltrate
0.4 ~ 0.8h of lower decolouring, filters to take filtrate, and filtrate finally is carried out into nanofiltration desalination；
7）Freeze-drying：20 ~ 35wt% polyethylene Topiramate Los oxazolidinones are added in enzymolysis liquid after nanofiltration desalination, freeze-drying obtains
Active peptides freeze-dried powder.Active peptides combination antigenic action immune cell activated, promotes it to be divided into the immune system of body
It is ripe, the activity and quantity of immunocyte are improved, strengthens itself antitumor ability.Above-mentioned active peptides are non-specific in reinforcement
Property it is immune while antitumor T lymphocyte specific immune response be present.Active peptides can suppress HL-60 and breed and lure
Guided cell apoptosis, its mechanism may be relevant with promoting the up-regulation of p53 protein expressions, Bcl-2 expression downward, but specifically mechanism and fails to understand
Really.
Embodiment 2：
The most preferred method of active peptides is prepared using the deep-sea fish flesh of fish, is comprised the following steps：
1）Degreasing：The deep-sea fish flesh of fish is cut into small pieces, and adds 3.2 ‰ chitosan-immobilized lipase, adds the distillation of 10 times of volumes
Water, constant temperature digests 1.4h at 30 DEG C, and enzymolysis terminates rear enzyme deactivation, is filtrated to get degreasing deep-sea fish flesh of fish degreasing.Chitosan is fixed
Change lipase preparation process be：With Chitosan powder is dried, with pH6.3 65mmol/L phosphate buffer soaked overnights,
Then the 1- ethyls -3- of Chitosan powder quality 1.0% is added（3- methylaminos）Propyl group carbodiimide activation 0.7h, is added
The glutaraldehyde coupling reaction 1.3h of 1.4 times of volumes of Chitosan powder, is cleaned 5 times with distilled water, and it is molten to add 1.2mg/mL lipase
Liquid and 0.3 ‰ bioactive peptides, are slowly stirred 1.1h in ice bath, then place refrigerator overnight, and secondary daily distilled water thoroughly rushes
Wash, filtration drying obtains chitosan-immobilized lipase.The amino acid sequence of bioactive peptide is
SCASVCKARCLRARGCRCVSVYCRCLR；
2）First time deodorant：The degreasing deep-sea fish flesh of fish is dipped in 0.6h in deodorant liquid, soaking temperature is 4 DEG C, deodorant liquid composition and
Its parts by weight is 0.7 part of 6 parts of Tea Polyphenols, 14 parts of pure water and grape seed extract；
3）Enzymolysis：The flesh of fish after first time deodorant is beaten into minced fillet, adds the distilled water of 3 times of volumes, add 0.2% it is thermophilic
Mycoproteinase and 0.4% chymotrypsin, digest 1.9h under 32 DEG C, natural ph, and 6 ‰ flavor proteases are added after enzyme deactivation,
In 52 DEG C, 0.7h is digested under the conditions of pH6.4, filters to take filtrate；
4）Second of deodorant：Saccharomyces cerevisiae and the Bifidobacterium of activation are inoculated with enzymolysis liquid, in:Ferment at constant temperature at 37 DEG C
2.2h.The addition of saccharomyces cerevisiae and Bifidobacterium is respectively 0.7 ‰ and 1.3 ‰；
5）Ultrafiltration：First using retention relative molecular mass 40kD film ultrafiltration, then retention relative molecular mass 5kD film is used to surpass
Filter；
6）Decolouring and desalination：1.1% activated carbon is added in ultrafiltrate, decolourize 0.6h under the conditions of 65 DEG C, rotating speed 150r/min,
Filtrate is filtered to take, filtrate is finally subjected to nanofiltration desalination；
7）Freeze-drying：30wt% polyethylene Topiramate Los oxazolidinones are added in enzymolysis liquid after nanofiltration desalination, freeze-drying obtains activity
More peptide freeze-dried powders.Active peptides combination antigenic action immune cell activated, promotes its differentiation and maturation in the immune system of body,
The activity and quantity of immunocyte are improved, strengthens itself antitumor ability.Above-mentioned active peptides are non-specific in reinforcement
Antitumor T lymphocyte specific immune response while immune be present.Active peptides can suppress HL-60 and breed and induce
Apoptosis, its mechanism may be relevant with promoting the up-regulation of p53 protein expressions, Bcl-2 expression downward, but specifically mechanism and fails to understand
Really.
Embodiment 3：
The method that active peptides are prepared using the deep-sea fish flesh of fish, is comprised the following steps：
1）Degreasing：The deep-sea fish flesh of fish is cut into small pieces, and adds 3 ‰ chitosan-immobilized lipase, adds the distilled water of 11 times of volumes,
Constant temperature digests 1.2h at 30 DEG C, and enzymolysis terminates rear enzyme deactivation, is filtrated to get degreasing deep-sea fish flesh of fish degreasing.Chitosan-immobilized fat
The preparation process of fat enzyme is：With Chitosan powder is dried, with pH6.5 70mmol/L phosphate buffer soaked overnights, then
Add the 1- ethyls -3- of Chitosan powder quality 0.9%（3- methylaminos）Propyl group carbodiimide activation 0.8h, adds shell and gathers
Icing Sugar end 1.3 times of volumes glutaraldehyde coupling reaction 1.2h, clean 6 times with distilled water, add 1.3mg/mL lipase solutions with
0.2 ‰ bioactive peptides, 1.0h is slowly stirred in ice bath, then places refrigerator overnight, secondary daily distilled water cleaning down,
Filtration drying obtains chitosan-immobilized lipase.The amino acid sequence of bioactive peptide is
SCASVCKARCLRARGCRCVSVYCRCLR；
2）First time deodorant：The degreasing deep-sea fish flesh of fish is dipped in 0.6h in deodorant liquid, soaking temperature is 4 DEG C, deodorant liquid composition and
Its parts by weight is 0.6 part of 6 parts of Tea Polyphenols, 16 parts of pure water and grape seed extract；
3）Enzymolysis：The flesh of fish after first time deodorant is beaten into minced fillet, adds the distilled water of 3 times of volumes, add 0.2% it is thermophilic
Mycoproteinase and 0.4% chymotrypsin, digest 1.9h under 32 DEG C, natural ph, and 6 ‰ flavor proteases are added after enzyme deactivation,
In 52 DEG C, 0.7h is digested under the conditions of pH6.4, filters to take filtrate；
4）Second of deodorant：Saccharomyces cerevisiae and the Bifidobacterium of activation are inoculated with enzymolysis liquid, in:Ferment at constant temperature at 37 DEG C
2.2h.The addition of saccharomyces cerevisiae and Bifidobacterium is respectively 0.7 ‰ and 1.3 ‰；
5）Ultrafiltration：First using retention relative molecular mass 40kD film ultrafiltration, then retention relative molecular mass 5kD film is used to surpass
Filter；
6）Decolouring and desalination：1.1% activated carbon is added in ultrafiltrate, decolourize 0.6h under the conditions of 65 DEG C, rotating speed 150r/min,
Filtrate is filtered to take, filtrate is finally subjected to nanofiltration desalination；
7）Freeze-drying：30wt% polyethylene Topiramate Los oxazolidinones are added in enzymolysis liquid after nanofiltration desalination, freeze-drying obtains activity
More peptide freeze-dried powders.Active peptides combination antigenic action immune cell activated, promotes its differentiation and maturation in the immune system of body,
The activity and quantity of immunocyte are improved, strengthens itself antitumor ability.Above-mentioned active peptides are non-specific in reinforcement
Antitumor T lymphocyte specific immune response while immune be present.Active peptides can suppress HL-60 and breed and induce
Apoptosis, its mechanism may be relevant with promoting the up-regulation of p53 protein expressions, Bcl-2 expression downward, but specifically mechanism and fails to understand
Really.
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 table
<110>The Gothic Bioisystech Co., Ltd of Lanxi City
<120>The method that active peptides are prepared using the deep-sea fish flesh of fish
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> PRT
<213>Artificial synthesized (Mytilus coruscus)
<400> 1
Ser Cys Ala Ser Val Cys Lys Ala Arg Cys Leu Arg Ala Arg Gly Cys
1 5 10 15
Arg Cys Val Ser Val Tyr Cys Arg Cys Leu Arg
20 25
Claims (10)
1. prepare the method for active peptides using the deep-sea fish flesh of fish, including it is degreasing, first time deodorant, enzymolysis, second of deodorant, super
Filter, simultaneously desalination and freeze-drying of decolourizing, it is characterised in that：Second described of deodorant step is the inoculation activation in enzymolysis liquid
Saccharomyces cerevisiae and Bifidobacterium, 1.5 ~ 3.5h of ferment at constant temperature at 32 ~ 43 DEG C.
2. the method according to claim 1 that active peptides are prepared using the deep-sea fish flesh of fish, it is characterised in that：Described is de-
Fat step is：The deep-sea fish flesh of fish is cut into small pieces, and adds 2 ~ 4 ‰ chitosan-immobilized lipase, adds the distillation of 8 ~ 15 times of volumes
Water, constant temperature digests 0.8 ~ 2.5h at 25 ~ 50 DEG C, and enzymolysis terminates rear enzyme deactivation, is filtrated to get the degreasing deep-sea fish flesh of fish.
3. the method according to claim 2 that active peptides are prepared using the deep-sea fish flesh of fish, it is characterised in that：Described shell
The preparation process of chitosan-immobilized lipase is：Chitosan powder is taken, with 60 ~ 80mmol/L phosphate-buffereds of pH6.0 ~ 6.8
Liquid soaked overnight, then add the 1- ethyls -3- of Chitosan powder quality 0.7 ~ 1.2%（3- methylaminos）Propyl group carbodiimide
0.5 ~ 0.9h is activated, the glutaraldehyde 1 ~ 1.8h of coupling reaction of 1 ~ 2 times of volume of Chitosan powder is added, 5 ~ 8 is cleaned with distilled water
It is secondary, 0.7 ~ 2.0mg/mL lipase solutions and 0.1 ~ 0.3 ‰ bioactive peptides are added, 0.5 ~ 1.5h are slowly stirred in ice bath, so
After place refrigerator overnight, secondary daily distilled water cleaning down, filtration drying obtains chitosan-immobilized lipase.
4. the method according to claim 3 that active peptides are prepared using the deep-sea fish flesh of fish, it is characterised in that：Described work
The amino acid sequence of property small peptide is SCASVCKARCLRARGCRCVSVYCRCLR.
5. the method according to claim 1 that active peptides are prepared using the deep-sea fish flesh of fish, it is characterised in that：Described
One time deodorant step is：The degreasing deep-sea fish flesh of fish is dipped in 0.4 ~ 0.8h in deodorant liquid, soaking temperature is 0 ~ 4 DEG C；The deodorant
Liquid composition and its parts by weight are 0.2 ~ 0.8 part of 3 ~ 8 parts of Tea Polyphenols, 10 ~ 20 parts of pure water and grape seed extract.
6. the method according to claim 1 that active peptides are prepared using the deep-sea fish flesh of fish, it is characterised in that：Described enzyme
Solving step is：The flesh of fish after first time deodorant is beaten into minced fillet, adds the distilled water of 2 ~ 4 times of volumes, adds 0.1 ~ 0.3%
Thermolysin and 0.2 ~ 0.5% chymotrypsin, in 27 ~ 40 DEG C, 1.5 ~ 3.0h is digested under natural ph, is added after enzyme deactivation
Enter 4 ~ 8 ‰ flavor proteases, in 45 ~ 55 DEG C, 0.5 ~ 1.0h is digested under the conditions of pH6.0 ~ 6.8, filters to take filtrate.
7. the method according to claim 1 that active peptides are prepared using the deep-sea fish flesh of fish, it is characterised in that：Described wine
The addition of brewer yeast and Bifidobacterium is respectively 0.5 ~ 1.0 ‰ and 0.8 ~ 1.8 ‰.
8. the method according to claim 1 that active peptides are prepared using the deep-sea fish flesh of fish, it is characterised in that：Described is super
Step is filtered first to use 30 ~ 40kD of retention relative molecular mass film ultrafiltration, then the film using 4 ~ 8kD of retention relative molecular mass
Ultrafiltration.
9. the method according to claim 1 that active peptides are prepared using the deep-sea fish flesh of fish, it is characterised in that：Described is de-
Simultaneously desalting steps are color：0.5 ~ 1.5% activated carbon is added in ultrafiltrate, under the conditions of 60 ~ 70 DEG C, 100 ~ 200r/min of rotating speed
Decolourized 0.4 ~ 0.8h, filters to take filtrate, and filtrate finally is carried out into nanofiltration desalination.
10. the method according to claim 1 that active peptides are prepared using the deep-sea fish flesh of fish, it is characterised in that：Described
Step is freeze-dried to add 20 ~ 35wt% polyethylene Topiramate Los oxazolidinones in the enzymolysis liquid after nanofiltration desalination, freeze-drying is lived
The more peptide freeze-dried powders of property.
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| CN108559764A (en) * | 2018-06-12 | 2018-09-21 | 艾苛密（上海）健康科技股份有限公司 | Anti-trioxypurine ocean fish oligopeptide and preparation method thereof |
| CN109097425A (en) * | 2018-08-28 | 2018-12-28 | 青海七彩花生物科技有限公司 | A kind of polypeptide and the purposes as influenza vaccines immunologic adjuvant |
| CN109673969A (en) * | 2018-12-27 | 2019-04-26 | 南京农业大学 | A kind of processing method of the quick-frozen barbecue taste processed meat products of formaldehydeless no pah4 |
| CN114532467A (en) * | 2022-03-16 | 2022-05-27 | 莱阳永安食品有限公司 | HPP (HPP) sterilization and preservation method of fruit juice |
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| CN1883289A (en) * | 2006-06-12 | 2006-12-27 | 湖北泰尔生物工程有限公司 | Method for preparing active polypeptide solution of fresh water fish protein |
| CN105614753A (en) * | 2015-12-28 | 2016-06-01 | 江瀚生物科技（上海）有限公司 | Micromolecule fish protein nutrition powder and manufacturing method thereof |
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| CN108294225A (en) * | 2018-01-29 | 2018-07-20 | 荣成鸿德海洋生物科技有限公司 | A kind of de- bitter fishy-removing-method of enzymolysis of fish proteins polypeptide liquid |
| CN108559764A (en) * | 2018-06-12 | 2018-09-21 | 艾苛密（上海）健康科技股份有限公司 | Anti-trioxypurine ocean fish oligopeptide and preparation method thereof |
| CN108559764B (en) * | 2018-06-12 | 2021-03-12 | 艾苛密（上海）健康科技股份有限公司 | Uric acid-reducing ocean fish oligopeptide and preparation method thereof |
| CN109097425A (en) * | 2018-08-28 | 2018-12-28 | 青海七彩花生物科技有限公司 | A kind of polypeptide and the purposes as influenza vaccines immunologic adjuvant |
| CN109097425B (en) * | 2018-08-28 | 2022-06-07 | 浙江康嘉基因技术有限公司 | Polypeptide and application of polypeptide as influenza vaccine immunologic adjuvant |
| CN109673969A (en) * | 2018-12-27 | 2019-04-26 | 南京农业大学 | A kind of processing method of the quick-frozen barbecue taste processed meat products of formaldehydeless no pah4 |
| CN109673969B (en) * | 2018-12-27 | 2022-01-04 | 南京农业大学 | Processing method of quick-frozen barbecue flavor conditioning meat product without formaldehyde and pah4 |
| CN114532467A (en) * | 2022-03-16 | 2022-05-27 | 莱阳永安食品有限公司 | HPP (HPP) sterilization and preservation method of fruit juice |
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