CN104910273B - A kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein, extraction and separation process - Google Patents
A kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein, extraction and separation process Download PDFInfo
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Abstract
The invention discloses a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein, extraction and separation process, steps are as follows: (1) chooses bivalve shellfish and temporarily support 2 ~ 7 days in clean sea water, reject dead shellfish；(2) induction is oriented after；(3) it opens shell and takes meat, be proportionally added into buffer solution, the heat-treated removal foreign protein of extracting solution；(4) sediment is removed through the screen to filtrate after extracting solution is cooling, supernatant integrates purification system by multi-ultrafiltration, micro-filtration, nanofiltration multicomponent membrane and removes macromolecular, separating-purifying and concentration, obtains MT crude product；(5) MT crude product obtains high-purity marine organisms source MT product by drying process through sephadex and ion-exchange chromatography united separation and purification.It so can effectively improve the intracorporal Metallothionein Content of shellfish, and can obtain to heavy metal with efficient removal effect, safer marine organisms source efficient heavy antidote product.
Description
Technical field
The present invention relates to the high-valued development technique fields of aquatic products, espespecially using ocean bivalve shellfish as raw material, through metal
The marine organisms source shellfish metallothionein that separation has efficient removal effect to heavy metal is extracted in induction.
Background technique
With the industrialized process in China, industrial and agricultural wastewater is largely discharged into natural water, causes immediate offshore area serious
Pollution, wherein heavy metal ion is extremely serious to water pollution.After environment is by heavy metal pollution, it is not only able to influence ocean
The physiological functions such as the ingesting of biology, growth and development and breeding, can also be enriched with, and enter human body structure by food chain in vivo
At potential threat, cause acute and chronic poisoning, the public hazards diseases such as " minamata disease ", " Itai-itai diseases ", " oligoleukocythemia " occurs, sternly
Jeopardize human health and safety again.Therefore, how to reduce heavy metal harm to the human body and have become current safety urgently to be resolved and ask
One of topic.
Heavy metal is drained very slowly naturally in vivo, is one of the essential measure of prevention and treatment poisoning using antidote,
But these antidotes mainly rely on chemical synthesis, often there is poorly water-soluble, bioavilability is low, and with different degrees of pair
Reaction.Therefore, that how to find a kind of efficiency natural there is the biological agent of detoxication just to seem rather important heavy metal.
Endogenous protein of the metallothionein (metallothionein, MT) as a kind of height inducibility, is mainly shown as in vivo
The storage and transportation of essential metal element is metabolized and fine-tunes and the removing toxic substances of nonessential metallic element, to protection brain, liver, kidney etc.
Vitals have meaning of crucial importance, medicine, cosmetics, food supplement, in terms of obtained it is extensive
Using.
Currently, MT majority common on domestic market is purified from rabbit liver, since MT raw materials requirement is special and related
The extractive technique of rabbit liver MT generallys use the works such as buffer extraction, organic solvent deposit removal of impurities, thermal denaturation removal of impurities, ethanol precipitation MT
Skill not only makes complex production process, there is no effective method energy mass production due to using a large amount of ethyl alcohol and chloroform in technique
MT causes the price of MT extremely expensive, and pollutes environment, so that its application and research be made to be restricted, MT raw material sources are
As one of the bottleneck for restricting field development.
MT resource abundant is selected, purification step is simplified, cost is reduced, especially reduces the use of toxic solvent, be raw
Produce the critical issue of MT.China is shellfish culture big country, the world, and marine shellfish cultured output accounts for the 80% of sea-farming total output,
Seashells industry is an important component of China's marine industries, but shellfish processing development rests on always primary rank
Section, even yield and the biggish main economic shellfish of export volume, the overwhelming majority also stops at the process segment of pre-treatment, shellfish
Based on the low freeze cutting product of added value, the intensely processed products of high added value are less for outlet.Bottom dwell Bivalve marine animal by
Have distribution wide in it, activity is low, has stronger bioaccumulation ability to heavy metal and is concerned, Bivalve marine animal
Internal MT content has certain correlation with Metals in Environments concentration, by exposed as instruction water environment pollution object and toxicity is imitated
Answer the major bio-markers of early warning.
Summary of the invention
The object of the present invention is to provide a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein,
Extraction and separation process can effectively improve the intracorporal Metallothionein Content of shellfish, and can obtain to heavy metal with efficient
Removing effect, safer marine organisms source efficient heavy antidote product.
To achieve the above object, solution of the invention is:
A kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein, extraction and separation process, step
Suddenly include:
Step 1, choose bivalve shellfish, using seawater clean the attached crop in shellfish surface, after in clean sea water temporarily support 2 ~ 7
It, keeps continuously logical oxygen, rejects dead shellfish during temporarily supporting in time；
After step 2, bivalve shellfish are temporarily supported, the seawater progress efficiently and directionally cultivated respectively in addition metal ion solution is lured
It leads, during which keeps natural lighting, the seawater of addition different metal ions solution, induction are replaced in continuous charge according to water quality situation
Time 1 ~ 30 day, ocean temperature maintained 10 ~ 25 DEG C；
Step 3, bivalve shellfish open shell and take meat, after homogenate, be directly entered the next step through metal directional induction；
Step 4, the shellfish meat through homogenized are proportionally added into buffer solution, adjust extracting solution pH to 6.0~9.5, control
Extracting temperature, stirring are extracted 0.5~6 hour；
The heat-treated removal foreign protein of step 5, extracting solution, is directly entered the next step；
Sediment is removed through the screen to filtrate after step 6, extracting solution are cooling, supernatant is more by multi-ultrafiltration, micro-filtration, nanofiltration
First film integrates purification system removing macromolecular, separating-purifying and concentration, obtains MT crude product；
Step 7, MT crude product are through sephadex and Hydrophobic Ionic displacement chromatography united separation and purification, at dry
Reason obtains high-purity marine organisms source MT product.
In the step 1, selection raw material is bivalve shellfish, comprising: oyster, mussel, scallop, clam.
In the step 2, the metal ion of addition is zinc salt, cadmium salt, mantoquita, chromic salts and the selenium salt of good water solubility；Addition
Concentration of metal ions be mass-volume concentration, i.e. mg/L, zinc ion concentration control in 0.5 ~ 40 mg/L, concentration of cadmium ions control
System is controlled in 0.02 ~ 1.3 mg/L, chromium ion concentration in 3 ~ 180 mg/L in 0.05 ~ 4 mg/L, copper ion concentration control, and selenium is dense
Degree control is in 0.04 ~ 2 mg/L.
In the step 2, metal inducement mode can be single metal inducement, can also mix simultaneously induction for various metals.
In the step 2, induction seawater salinity maintains 15 ~ 35 ‰, and induction seawater pH control is 6.5 ~ 8.5.
In the step 3, shellfish opening uses high temperature gas flow or freezing processing method.
In the step 4, through being homogenized the mass volume ratio of shellfish meat and buffer solution, i.e., kilogram: rising, be 1:2~1:10.
In the step 4, the buffer solution that uses for the Tris-HCL of 0.01~0.3mol/L or phosphate buffer solution or
Sodium chloride solution, Extracting temperature are controlled at 20~60 DEG C.
In the step 5, extracting solution is 60 DEG C by heating removal part foreign protein, heating temperature, and the time controls 30
~60 minutes.
In the step 6, after 150 μm of the screen to filtrates, the ceramic membrane or plate membrane for being directly over 0.5um carry out extracting solution
Filtering clarification, then the rolled film through molecular cut off 50KD, 5KD and 1KD carry out multi-stage separation.
In the step 7, MT crude product through sephadex G 75 and Hydrophobic Ionic displacement chromatography united separation and purification, after
It is dried, obtains high-purity marine organisms source MT product.
After adopting the above scheme, the MT that the present invention uses induces extraction process, changes in the past with animal's livers such as rabbit livers
The use of a large amount of organic solvent ethyl alcohol and chloroform and the limitation of raw material sources when preparing MT for raw material, not only reduce because
It adds these reagents and causes the influence of safety, and reduce removal technique subsequent for reagent, and select abundant double
Shell shellfish is used as MT raw material sources, reduces its industry cost.
In addition, the present invention establishes marine organisms source MT industrialized technology, it is prepared for relying primarily on chemistry compared to current
Synthesis, there are poorly water-solubles, and bioavilability is low, and aggravate with subcutaneous purple plague purpura, nose bleeding gums, abdominal distension nausea, bicker gruel
The heavy metal detoxification agent of the side reactions such as rotten, fever fash, obtains safer efficient marine organisms source heavy metal removing agent and produces
Product change simultaneously pattern of the shellfish outlet based on freezing, increase added value of product, and promoting the high level of Mollusca Resource to develop has
Vital effect, shellfish MT by be the following MT industry development an important directions.
Specific embodiment
Present invention discloses a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein, extracts and divide
Separating process, step include:
Step 1, choose bivalve shellfish, using seawater clean the attached crop in shellfish surface, after in clean sea water temporarily support 2 ~ 7
It, keeps continuously logical oxygen, rejects dead shellfish during temporarily supporting in time；The bivalve shellfish is oyster, mussel, scallop or clam
Deng；
After step 2, bivalve shellfish are temporarily supported, cultivates contain zinc salt, cadmium salt, mantoquita, chromic salts or selenium salt metal in addition respectively
The seawater of solion carries out efficiently and directionally induction, and the addition manner of metal salt is single metal salt or various metals salt, addition
Zinc ion concentration control in 0.5 ~ 40 mg/L, concentration of cadmium ions control exists in 0.05 ~ 4 mg/L, copper ion concentration control
During which 0.02 ~ 1.3 mg/L, chromium ion concentration control keep nature in 0.04 ~ 2 mg/L in 3 ~ 180 mg/L, selenium concentration control
The seawater of addition different metal ions solution is replaced in illumination, continuous charge according to water quality situation, and the metal ion of addition is water-soluble
Property good zinc salt, cadmium salt, mantoquita, chromic salts and selenium salt；The concentration of metal ions of addition is mass-volume concentration, i.e. milligram: being risen
(mg/L), zinc ion concentration control is in 0.5 ~ 40 mg/L, and concentration of cadmium ions control is in 0.05 ~ 4 mg/L, copper ion concentration control
In 0.02 ~ 1.3 mg/L, chromium ion concentration control in 3 ~ 180 mg/L, selenium concentration control in 0.04 ~ 2 mg/, induction time 1 ~
30 days, ocean temperature maintained 10 ~ 25 DEG C, and induction seawater salinity maintains 15 ~ 35 ‰, and induction seawater pH control is 6.5 ~ 8.5；Gold
Belonging to induction mode can be single metal inducement, can also mix simultaneously induction for various metals；
Step 3, bivalve shellfish open shell and take meat, after homogenate, be proportionally added into buffer solution through metal directional induction, adjust
Extracting solution pH to 6.0~9.5, controls Extracting temperature, and stirring is extracted 0.5~6 hour；Through the quality for being homogenized shellfish meat and buffer solution
Volume ratio, i.e., kilogram: rising, be 1:2~1:10；The buffer solution used is that the Tris-HCL of 0.01~0.3mol/L or phosphoric acid are slow
Solution or sodium chloride solution are rushed, control extraction process pH is 6.0~9.5, and at 20~60 DEG C, time control exists for Extracting temperature control
0.5~6 hour；Shellfish opening uses high temperature gas flow or freezing processing method；
The heat-treated removal foreign protein of step 4, extracting solution, is directly entered the next step；Extracting solution can pass through heating
Part foreign protein is removed, heating temperature is 60 DEG C, and the time controlled at 30~60 minutes, and in a small amount of extraction process, 80 DEG C add
Heat 5~10 minutes, also part that can be removed foreign protein, it is contemplated that be difficult to a tonne extracting solution in the short time in actual industrial production
The interior control for carrying out heating and cooling, heating temperature are preferably controlled at 60 DEG C；
Bulky grain sediment is removed through 150 μm of sieves after step 5, extracting solution are cooling, extracting solution after filtering, is directly over
The ceramic membrane or plate membrane of 0.5um is filtered clarification, then the rolled film through molecular cut off 50KD, 5KD and 1KD carries out multistage
Separating-purifying and concentration are separated, MT crude product is obtained；
Step 6, MT crude product are isolated and purified through sephadex and Hydrophobic Ionic displacement chromatography filler, at dry
Reason obtains high-purity marine organisms source MT product.
The present invention is illustrated below by way of specific implementation case, the advantages and features of the present invention will with description and more
It is clear.But these case study on implementation be only it is exemplary, it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that without departing from the spirit and scope of the invention can details to inventive technique scheme and form carry out
Modifications or substitutions, but these modifications and replacement each fall within protection scope of the present invention.
Embodiment one:
Choose bivalve shellfish oyster, using seawater clean the attached crop in shellfish surface, after in clean sea water temporarily support 7 days, protect
Continuously logical oxygen is held, rejects dead shellfish during temporarily supporting in time.After bivalve shellfish is temporarily supported, cultivates contain 40 mg/L in addition respectively
The seawater of zinc salt carries out efficiently and directionally induction, during which keeps natural lighting, and continuous charge replaces addition metal according to water quality situation
The seawater of solion, induction time 20 days, ocean temperature maintained 20 DEG C, and induction seawater salinity maintains 28 ‰, induced seawater
PH is controlled 8.2.Bivalve shellfish opens shell and takes meat through metal directional induction, after homogenate, weighs homogenate raw material 50Kg, by raw material and
The mass volume ratio of buffer solution is 1:4,0.25 mol/L Tris-HCL buffer of 200L is put into, with hydrochloric acid and sodium hydroxide
Extracting solution pH to 9.0 is controlled, 30 DEG C of stirrings are extracted 4 hours, and extracting solution is through 60 DEG C of heating, 30 minutes removal foreign proteins.Extract liquid cooling
But bulky grain sediment is removed through 150 μm of sieves afterwards, extracting solution after filtering, is directly over the ceramic membrane or plate membrane of 0.5um
It is filtered clarification, then the rolled film through molecular cut off 50KD, 5KD and 1KD carries out multi-stage separation separating-purifying and concentration, obtains
Obtain MT crude product.MT crude product is isolated and purified through sephadex G -75 and Hydrophobic Ionic displacement chromatography filler, by freeze-drying
Processing obtains high-purity marine organisms source MT product.
Embodiment two:
Choose bivalve shellfish scallop, using seawater clean the attached crop in shellfish surface, after in clean sea water temporarily support 5 days, protect
Continuously logical oxygen is held, rejects dead shellfish during temporarily supporting in time.After bivalve shellfish is temporarily supported, cultivates contain 0.5mg/L in addition respectively
The seawater of cadmium salt carries out efficiently and directionally induction, during which keeps natural lighting, and continuous charge replaces addition metal according to water quality situation
The seawater of solion, induction time 10 days, ocean temperature maintained 18 DEG C, and induction seawater salinity maintains 26 ‰, induced seawater
PH is controlled 8.2.Bivalve shellfish opens shell and takes meat through metal directional induction, after homogenate, weighs homogenate raw material 50Kg, by raw material and
The mass volume ratio of buffer solution is 1:10,0.05 mol/L sodium chloride buffer of 500L is put into, with hydrochloric acid and sodium hydroxide
Extracting solution pH to 7.5 is controlled, 45 DEG C of stirrings are extracted 2 hours, and extracting solution is through 60 DEG C of heating, 60 minutes removal foreign proteins.Extract liquid cooling
But bulky grain sediment is removed through 150 μm of sieves afterwards, extracting solution after filtering, is directly over the ceramic membrane or plate membrane of 0.5um
It is filtered clarification, then the rolled film through molecular cut off 50KD, 5KD and 1KD carries out multi-stage separation separating-purifying and concentration, obtains
Obtain MT crude product.MT crude product is isolated and purified through sephadex G -75 and Hydrophobic Ionic displacement chromatography filler, by freeze-drying
Processing obtains high-purity marine organisms source MT product.
Embodiment three:
Choose bivalve shellfish clam, using seawater clean the attached crop in shellfish surface, after in clean sea water temporarily support 3 days, protect
Continuously logical oxygen is held, rejects dead shellfish during temporarily supporting in time.After bivalve shellfish is temporarily supported, cultivates contain 0.3mg/L in addition respectively
The seawater of mantoquita carries out efficiently and directionally induction, during which keeps natural lighting, and continuous charge replaces addition metal according to water quality situation
The seawater of solion, induction time 30 days, ocean temperature maintained 22 DEG C, and induction seawater salinity maintains 30 ‰, induced seawater
PH is controlled 8.5.Bivalve shellfish opens shell and takes meat through metal directional induction, after homogenate, weighs homogenate raw material 50Kg, by raw material and
The mass volume ratio of buffer solution is 1:5, puts into 0.15 mol/L phosphate buffer of 250L, is controlled with hydrochloric acid and sodium hydroxide
Extracting solution pH to 8.5,55 DEG C of stirrings are extracted 1 hour, and extracting solution is through 60 DEG C of heating, 30 minutes removal foreign proteins.After extracting solution is cooling
Bulky grain sediment is removed through 150 μm of sieves, after filtering, the ceramic membrane or plate membrane for being directly over 0.5um carry out extracting solution
Filtering clarification, then the rolled film through molecular cut off 50KD, 5KD and 1KD carry out multi-stage separation separating-purifying and concentration, obtain MT
Crude product.MT crude product is isolated and purified through sephadex G -75 and Hydrophobic Ionic displacement chromatography filler, by freeze-drying process,
Obtain high-purity marine organisms source MT product.
Example IV:
Choose bivalve shellfish mussel, using seawater clean the attached crop in shellfish surface, after in clean sea water temporarily support 4 days, protect
Continuously logical oxygen is held, rejects dead shellfish during temporarily supporting in time.After bivalve shellfish is temporarily supported, cultivates contain 23mg/L chromium in addition respectively
The seawater of salt carries out efficiently and directionally induction, during which keeps natural lighting, continuous charge, according to water quality situation replace addition metal from
The seawater of sub- solution, induction time 7 days, ocean temperature maintained 10 DEG C, and induction seawater salinity maintains 25 ‰, induction seawater pH control
System is 7.5.Bivalve shellfish opens shell and takes meat through metal directional induction, after homogenate, homogenate raw material 50Kg is weighed, by raw material and buffering
The mass volume ratio of solution is 1:8, puts into 0.10 mol/L Tris-HCL buffer of 400L, is controlled with hydrochloric acid and sodium hydroxide
Extracting solution pH to 7.5,45 DEG C of stirrings are extracted 3 hours, and extracting solution is through 60 DEG C of heating, 40 minutes removal foreign proteins.After extracting solution is cooling
Bulky grain sediment is removed through 150 μm of sieves, after filtering, the ceramic membrane or plate membrane for being directly over 0.5um carry out extracting solution
Filtering clarification, then the rolled film through molecular cut off 50KD, 5KD and 1KD carry out multi-stage separation separating-purifying and concentration, obtain MT
Crude product.MT crude product is isolated and purified through sephadex G -75 and Hydrophobic Ionic displacement chromatography filler, by freeze-drying process,
Obtain high-purity marine organisms source MT product.
Embodiment five:
Choose bivalve shellfish oyster, using seawater clean the attached crop in shellfish surface, after in clean sea water temporarily support 7 days, protect
Continuously logical oxygen is held, rejects dead shellfish during temporarily supporting in time.After bivalve shellfish is temporarily supported, cultivates contain 0.5mg/L in addition respectively
The seawater of selenium salt carries out efficiently and directionally induction, during which keeps natural lighting, and continuous charge replaces addition metal according to water quality situation
The seawater of solion, induction time 10 days, ocean temperature maintained 15 DEG C, and induction seawater salinity maintains 18 ‰, induced seawater
PH is controlled 7.0.Bivalve shellfish opens shell and takes meat through metal directional induction, after homogenate, weighs homogenate raw material 50Kg, by raw material and
The mass volume ratio of buffer solution is 1:4, and investment 200L 0.1mol/L phosphate buffer (it is molten to include 0.1 mol/L sodium chloride
Liquid), extracting solution pH to 9.2 is controlled with hydrochloric acid and sodium hydroxide, 45 DEG C of stirrings are extracted 6 hours, and extracting solution heats 30 points through 60 DEG C
Clock removes foreign protein.Bulky grain sediment is removed through 150 μm of sieves after extracting solution is cooling, extracting solution after filtering, is directly over
The ceramic membrane or plate membrane of 0.5um is filtered clarification, then the rolled film through molecular cut off 50KD, 5KD and 1KD carries out multistage
Separating-purifying and concentration are separated, MT crude product is obtained.MT crude product is through sephadex G -75 and Hydrophobic Ionic displacement chromatography filler
It isolates and purifies, by freeze-drying process, obtains high-purity marine organisms source MT product.
Embodiment six:
Choose bivalve shellfish oyster, using seawater clean the attached crop in shellfish surface, after in clean sea water temporarily support 7 days, protect
Continuously logical oxygen is held, rejects dead shellfish during temporarily supporting in time.After bivalve shellfish is temporarily supported, cultivates contain 4mg/L zinc in addition respectively
Salt, 0.5mg/L cadmium salt, 0.2mg/L mantoquita, 2mg/L selenium salt and 12mg/L chromic salts seawater to carry out various metals efficient joint fixed
To induction, natural lighting is during which kept, the seawater of addition metal ion solution, induction are replaced in continuous charge according to water quality situation
Time 15 days, ocean temperature maintained 20 DEG C, and induction seawater salinity maintains 28 ‰, and induction seawater pH control is 8.0.Bivalve shellfish
It through metal directional induction, opens shell and takes meat, after homogenate, homogenate raw material 50Kg is weighed, by the mass volume ratio of raw material and buffer solution
For 1:4,200L 0.1mol/L Tris-HCL buffer is put into, controls extracting solution pH to 9.2 with hydrochloric acid and sodium hydroxide, 40 DEG C
Stirring is extracted 4 hours, and extracting solution is through 60 DEG C of heating, 30 minutes removal foreign proteins.It is big through 150 μm of sieve removals after extracting solution is cooling
Particle sediment, after filtering, the ceramic membrane or plate membrane for being directly over 0.5um are filtered clarification to extracting solution, then through retaining
The rolled film of molecular weight 50KD, 5KD and 1KD carry out multi-stage separation separating-purifying and concentration, obtain MT crude product.MT crude product is poly- through Portugal
Sugared gel G-75 and Hydrophobic Ionic displacement chromatography filler isolate and purify, and by freeze-drying process, it is raw to obtain high-purity ocean
Material resource MT product.
Embodiment seven:
Choose bivalve shellfish scallop, using seawater clean the attached crop in shellfish surface, after in clean sea water temporarily support 7 days, protect
Continuously logical oxygen is held, rejects dead shellfish during temporarily supporting in time.After bivalve shellfish is temporarily supported, cultivates contain 4mg/L zinc in addition respectively
The seawater of salt and 1.0mg/L cadmium salt carries out two kinds of metal efficient joint directional inductions, during which holding natural lighting, continuous charge,
The seawater of addition metal ion solution is replaced according to water quality situation, induction time 14 days, ocean temperature maintained 20 DEG C, induced seawater
Salinity maintains 32 ‰, and induction seawater pH control is 8.5.Bivalve shellfish opens shell and takes meat through metal directional induction, after homogenate, claims
Homogenate raw material 50Kg is taken, is 1:7 by the mass volume ratio of raw material and buffer solution, puts into 350L 0.25mol/L Tris-HCL
Buffer controls extracting solution pH to 8.6 with hydrochloric acid and sodium hydroxide, and 45 DEG C of stirrings are extracted 2 hours, and extracting solution is through 60 DEG C of heating 30
Minute removal foreign protein.Bulky grain sediment is removed through 150 μm of sieves after extracting solution is cooling, extracting solution after filtering, directly passes through
The ceramic membrane or plate membrane for crossing 0.5um are filtered clarification, then the progress of the rolled film through molecular cut off 50KD, 5KD and 1KD is more
Grade separation separating-purifying and concentration, obtain MT crude product.MT crude product is filled out through sephadex G -75 and Hydrophobic Ionic displacement chromatography
Material isolates and purifies, and by freeze-drying process, obtains high-purity marine organisms source MT product.
Embodiment eight:
Choose bivalve shellfish mussel, using seawater clean the attached crop in shellfish surface, after in clean sea water temporarily support 7 days, protect
Continuously logical oxygen is held, rejects dead shellfish during temporarily supporting in time.After bivalve shellfish is temporarily supported, cultivates contain 0.4mg/L in addition respectively
The seawater of selenium salt and 1.0mg/L cadmium salt carries out two kinds of metal efficient joint directional inductions, during which keeps natural lighting, continuously fills
Gas replaces the seawater of addition metal ion solution according to water quality situation, and induction time 10 days, ocean temperature maintained 15 DEG C, induction
Seawater salinity maintains 28 ‰, and induction seawater pH control is 8.0.Bivalve shellfish opens shell and takes meat through metal directional induction, homogenate
Afterwards, homogenate raw material 50Kg is weighed, is 1:4 by the mass volume ratio of raw material and buffer solution, puts into 200L 0.15mol/L phosphoric acid
Buffer controls extracting solution pH to 8.0 with hydrochloric acid and sodium hydroxide, and 35 DEG C of stirrings are extracted 6 hours, and extracting solution is through 60 DEG C of heating 30
Minute removal foreign protein.Bulky grain sediment is removed through 150 μm of sieves after extracting solution is cooling, extracting solution after filtering, directly passes through
The ceramic membrane or plate membrane for crossing 0.5um are filtered clarification, then the progress of the rolled film through molecular cut off 50KD, 5KD and 1KD is more
Grade separation separating-purifying and concentration, obtain MT crude product.MT crude product is filled out through sephadex G -75 and Hydrophobic Ionic displacement chromatography
Material isolates and purifies, and by freeze-drying process, obtains high-purity marine organisms source MT product.
Embodiment nine:
Choose bivalve shellfish oyster, using seawater clean the attached crop in shellfish surface, after in clean sea water temporarily support 7 days, protect
Continuously logical oxygen is held, rejects dead shellfish during temporarily supporting in time.After bivalve shellfish is temporarily supported, cultivates contain 2.5mg/L in addition respectively
The seawater of zinc salt, 2.0mg/L selenium salt and 24mg/L chromic salts carries out two kinds of metal efficient joint directional inductions, during which keeps natural light
According to the seawater of addition metal ion solution is replaced in continuous charge according to water quality situation, and induction time 15 days, ocean temperature maintained
15 DEG C, induction seawater salinity maintains 18 ‰, and induction seawater pH control is 6.8.Bivalve shellfish is opened shell and is taken through metal directional induction
Meat after homogenate, weighs homogenate raw material 50Kg, is 1:2 by the mass volume ratio of raw material and buffer solution, puts into 100L 0.3mol/
L sodium chloride buffer controls extracting solution pH to 9.0 with hydrochloric acid and sodium hydroxide, and 30 DEG C of stirrings are extracted 6 hours, and extracting solution is through 60
DEG C heating 60 minutes removal foreign proteins.Bulky grain sediment is removed through 150 μm of sieves after extracting solution is cooling, extracting solution is through filtering
Afterwards, the ceramic membrane or plate membrane for being directly over 0.5um are filtered clarification, then the volume through molecular cut off 50KD, 5KD and 1KD
Formula film carries out multi-stage separation separating-purifying and concentration, obtains MT crude product.MT crude product is through sephadex G -75 and Hydrophobic Ionic
Displacement chromatography filler isolates and purifies, and by freeze-drying process, obtains high-purity marine organisms source MT product.
Using cheap, abundant marine fishery shellfish simplifies purification procedures, provides a kind of high-purity the present invention as raw material
The extraction purification separating technology of marine organisms source MT.MT extraction process disclosed by the invention, which changes, prepared MT in the past with rabbit liver
When a large amount of organic solvent ethyl alcohol and chloroform use, not only reduce the influence for causing safety because adding these reagents, and
And removal technique subsequent for reagent is reduced, its industry cost is reduced, while using Mollusca Resource abundant for MT
Raw material overcomes the raw material sources problem for restricting MT industry development, and promotes the intensive processing of shellfish high value-added industries.It compares
Chemical synthesis is relied primarily at present, there are poorly water-solubles, and bioavilability is low, and aggravated with subcutaneous purple plague purpura, nose bleeding gums,
Abdominal distension nausea, perleche, the fever side reactions such as fash heavy metal detoxification agent, by the technique can obtain high added value,
Wide market, safer efficient marine organisms source heavy metal removing agent product.
It should be noted that above-mentioned case study on implementation is only not limit this hair to preferable case study on implementation of the invention
Bright protection scope, made equivalent replacement or substitution belong to protection model of the invention based on the above technical solution
It encloses, protection scope of the present invention is subject to claims.
Claims (10)
1. a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein, extraction and separation process, step
Include:
Step 1, choose bivalve shellfish, using seawater clean the attached crop in shellfish surface, after in clean sea water temporarily support 2 ~ 7 days, protect
Continuously logical oxygen is held, rejects dead shellfish during temporarily supporting in time；
After step 2, bivalve shellfish are temporarily supported, the seawater cultivated respectively in addition metal ion solution carries out efficiently and directionally induction, phase
Between keep natural lighting, the seawater of addition different metal ions solution is replaced in continuous charge according to water quality situation, induction time 1 ~
30 days, ocean temperature maintained 10 ~ 25 DEG C；
Step 3, bivalve shellfish open shell and take meat, after homogenate, be directly entered the next step through metal directional induction；
Step 4, the shellfish meat through homogenized are proportionally added into buffer solution, adjust extracting solution pH to 6.0~9.5, and control is extracted
Temperature, stirring are extracted 0.5~6 hour；
The heat-treated removal foreign protein of step 5, extracting solution, is directly entered the next step；
Sediment is removed through the screen to filtrate after step 6, extracting solution are cooling, supernatant passes through multi-ultrafiltration, micro-filtration, nanofiltration multicomponent membrane
Integrated purification system removing macromolecular, separating-purifying and concentration, obtain metallothionein crude product；
Step 7, metallothionein crude product are through sephadex and Hydrophobic Ionic displacement chromatography united separation and purification, by drying
Processing obtains high-purity marine organisms source metallothionein product.
2. a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein as described in claim 1 mentions
Take separating technology, it is characterised in that: in the step 1, selection raw material is bivalve shellfish, comprising: oyster, mussel, scallop, clam
Clam.
3. a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein as described in claim 1 mentions
Take separating technology, it is characterised in that: in the step 2, the metal ion of addition is zinc salt, cadmium salt, mantoquita, the chromium of good water solubility
Salt and selenium salt；The concentration of metal ions of addition is mass-volume concentration, i.e. mg/L, zinc ion concentration control in 0.5 ~ 40 mg/L,
Concentration of cadmium ions control in 0.05 ~ 4 mg/L, copper ion concentration control in 0.02 ~ 1.3 mg/L, chromium ion concentration control 3 ~
180 mg/L, selenium concentration are controlled in 0.04 ~ 2 mg/L.
4. a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein as claimed in claim 1 or 2,
Extraction and separation process, it is characterised in that: in the step 2, metal inducement mode be single metal inducement or various metals simultaneously
Mixing induction.
5. a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein as described in claim 1 mentions
Take separating technology, it is characterised in that: in the step 2, induction seawater salinity maintains 15 ~ 35 ‰, and induction seawater pH control exists
6.5~8.5。
6. a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein as described in claim 1 mentions
It takes separating technology, it is characterised in that: in the step 4, through being homogenized the mass volume ratio of shellfish meat and buffer solution, i.e., kilogram: rising,
For 1:2~1:10.
7. a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein as described in claim 1 or 6,
Extraction and separation process, it is characterised in that: in the step 4, the buffer solution used is the Tris-HCL of 0.01~0.3mol/L
Or phosphate buffer solution or sodium chloride solution, Extracting temperature are controlled at 20~60 DEG C.
8. a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein as described in claim 1 mentions
Take separating technology, it is characterised in that: in the step 5, extracting solution passes through heating removal part foreign protein, heating temperature 60
DEG C, the time controlled at 30~60 minutes.
9. a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein as described in claim 1 mentions
Take separating technology, it is characterised in that: in the step 6, extracting solution is directly over the ceramics of 0.5um after 150 μm of the screen to filtrates
Film or plate membrane are filtered clarification, then the rolled film through molecular cut off 50KD, 5KD and 1KD carries out multi-stage separation.
10. a kind of induction of marine organisms source efficient heavy scavenger shellfish metallothionein as described in claim 1 mentions
Take separating technology, it is characterised in that: in the step 7, metallothionein crude product is handed over through sephadex G 75 and Hydrophobic Ionic
Chromatography united separation and purification is changed, by drying process, obtains high-purity marine organisms source metallothionein product.
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| In situ potential use of metallothionein as a biomarker of cadmium contamination in Ruditapes decussatus.;W.Smaoui-Damak et al.;《Ecotoxicology and Environmental Safety》;20091231;1489-1498 |
| 金属离子对水生动物体内金属硫蛋白应激诱导研究进展;李诗逸 等;《水产科学》;20140930;第33卷(第9期);594-600 |
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