CN109628418A - A kind of marine organisms source superoxide dismutase extraction and separation process - Google Patents
A kind of marine organisms source superoxide dismutase extraction and separation process Download PDFInfo
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- C12Y115/01001—Superoxide dismutase (1.15.1.1)
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Abstract
The invention discloses a kind of marine organisms source superoxide dismutase extraction and separation process, a kind of marine organisms source superoxide dismutase extraction and separation process of the invention, it carry out be after coarse extraction by internal organ and fish blood system, the extracting solution obtained again to centrifugation purifies, extract the superoxide dismutase of preparation, purity is high, yield are high, and unit enzyme activity can achieve 6000U/mg or more.In the use for carrying out changing a large amount of organic solvent and inorganic salts sulphur ammonium in traditional extraction process when internal organ coarse extraction and fish blood coarse extraction, it not only reduces because of the influence caused by adding these reagents to superoxide dismutase activity and safety, and waste and subsequent removal technique for reagent are reduced, reduce its industry cost.Raw material are the internal organ and fish blood of ocean fish, effectively reduce because processing fishery -ies product generates environmental pollution and the wasting of resources brought by a large amount of by-products, realize waste utilization, turn waste into wealth.
Description
Technical field
The present invention relates to superoxide dismutase technical field of traction and preparation, specifically a kind of marine organisms source superoxides
Mutase extraction and separation process.
Background technique
Superoxide dismutase, alias orgotein, abbreviation: SOD.SOD is a kind of active material derived from life entity, can be disappeared
The harmful substance generated in metabolic processes except organism.The special effect that SOD has anti-aging is constantly supplemented human body
Fruit.Superoxide dismutase is to count isolated superoxide dismutase since ox red blood cell for the first time for 1938, people
To SOD research oneself have more than 70 years history.McCord in 1969 etc. rediscovers this albumen, and it was found that they
Bioactivity has understood fully that it is catalyzed the property that disproportionated reaction occurs for superoxide anion, so being formally named as superoxides
Mutase.
Superoxide dismutase is a kind of novel enzyme preparation.It is wide in the distributed pole of living nature, almost from animal to plant,
Even from people to unicellular organism, there is its presence.SOD is considered as in Life Science in the enzyme, human body of most magical magic power
Rubbish street cleaner.SOD is the natural enemy of oxygen radical, is the number one killer of oxygen radical in body, be life and health it
This.SOD is antioxidase important in organism, is distributed widely in various organisms, such as animal, plant, microorganism etc..
SOD has special physiological activity, is the primary substance that free radical is removed in organism.The horizontal height of SOD in vivo
Mean aging and dead intuitive index；Now it has proven convenient that up to more than 60 kinds of disease caused by oxygen radical.It can fight with
It blocks because oxygen radical is damaging caused by cell, and repairs damaged cell in time, cell is hurt caused by restoring because of free radical
Evil.Due to modern life pressure, environmental pollution, various radiation and excess movement can all cause oxygen radical largely to be formed；Therefore,
The status of SOD is more and more important in biological antioxidant mechanism！
China's traditional industry is extracted from the blood of animal and internal organs mostly at present, but this kind of raw material sources are limited,
Complicated component, and storage, in terms of there are inconveniences, therefore production technology is easy to cause to complicate, makes to be produced into
This substantial increase.It is built so far in terms of SOD rule of origin mainly or based on the animal tissues such as pigs and cattle blood, liver
A variety of methods that SOD is isolated and purified from animal erythrocyte are found.During the extraction purification of traditional SOD, use respectively
A large amount of organic solvent-acetone and inorganic salts sulfuric acid are pressed, and the addition of these reagents not only influences the activity of enzyme, but also for examination
The waste of agent and subsequent removal technique, improve its industry cost.And animal itself carries various bacterial viruses etc., this
Just inevitably it is easy to happen some cross-infections, phenomena such as allergic reaction, especially aftosa, rabid ox disease and by moving
The sprawling for the pernicious infectious disease that object is propagated, people produce query for the safety for extracting SOD isoreactivity ingredient from pig, ox,
The increased risk of animal sources blood product is produced, many countries and regions are resisted using animal blood and internal organ as the super of waste
Superoxide dismutase, so that superoxide dismutase is extremely in short supply in the international market.
Therefore it needs to propose a kind of using marine organisms as the superoxide dismutase extraction and separation process of raw material.
Summary of the invention
The purpose of the present invention is to provide a kind of marine organisms source superoxide dismutase extraction and separation process, on solving
State the problem of proposing in background technique.
To achieve the above object, the invention provides the following technical scheme:
A kind of marine organisms source superoxide dismutase extraction and separation process, specifically includes the following steps:
S1, it takes fresh ocean fish, takes out internal organ and fish blood, internal organ are cleaned up, drain rear spare, be added into fish blood
The sodium citrate of 5-10% fish blood quality, refrigerates spare after mixing；
S2, the internal organ cleaned up are beaten, obtain internal organ slurries；
S3, phosphate buffer solution is added into internal organ slurries, adjusts extracting solution pH6.5-7.5, stirs and extract at 40-60 DEG C
2-6h obtains internal organ extracting solution；
S4, heating removal foreign protein is carried out to internal organ extracting solution, rear centrifugation removal precipitating obtains supernatant A；
S5, fish blood is placed in a centrifuge to centrifugation, red blood cell is made in sediment brine, broken with ultrasonic wave
Wall processing is added deionized water, obtains hemolysate after standing under ice bath；
S6, the ethanol solution that 0-4 DEG C is added into hemolysate, add 0-4 DEG C of chloroform, then stir, stand, centrifugation
Supernatant is collected afterwards and obtains crude enzyme liquid, acetone is added into crude enzyme liquid, is then stirred, is centrifuged, taking precipitate；
S7, the obtained sediment of step S6 is dissolved with phosphate buffer, stir, be centrifuged after obtain supernatant, pass through
Dialysis removes salt ion, obtains supernatant C；
S8, merge supernatant A and supernatant C, obtain mixed liquor, mixed liquor is concentrated by ultrafiltration, obtain concentrate, it will
Concentrate heat temperature raising, adjusting pH value are 8.5-9.0, and add protective agent heat preservation, cooling after heat preservation, and centrifugation removal precipitating obtains
Supernatant D；
S9, supernatant D is dissolved in the deionized water containing ethyl alcohol, the thick solution of SOD is obtained, with the stream of 30-70ml/h
Speed makes the thick solution of SOD pass through modified chromatographic column, then the remaining thick solution of SOD in modified chromatographic column is washed with deionized water；
S10, the thick solution of SOD adsorbed with ethyl alcohol and sodium-chloride water solution elution macroporous weak base ion exchange resin, are collected
Efflux is concentrated, is freeze-dried up to the superoxide dismutase purified.
Further: in the step S3, the additional amount of phosphate buffer solution is that 6- is added in every kilogram of internal organ slurries
10L。
Further: in the step S4, extracting solution is 60-80 DEG C by heating removal part foreign protein, heating temperature,
Heating time is 60-140min.
Further: in the step S5, the ultrasonic power of supersonic wave wall breaking processing is 150-250W, and ultrasonic time is
10-25min。
Further: in the step S6, the additional amount of ethanol solution is the volume of 20-40% hemolysate, and chloroform adds
Enter the volume that amount is 15-25% hemolysate, the additional amount of acetone is 3-4 times of volume of crude enzyme liquid.
Further: in the step S7, the revolving speed of centrifuge is 3000-5000r/min, centrifugation time 20-
30min。
Further: in the step S8, protective agent is lauroyl chloride, and the additive amount of lauroyl chloride is concentrate quality
0.02-0.04%；Soaking time is 2-3h, is cooled to 10-20 DEG C after heat preservation, and supernatant is collected by centrifugation, and wherein centrifugal speed is
5000-8000r/min。
It is further: in the step S9, modified chromatographic column the preparation method comprises the following steps: take macroporous weak base ion exchange resin,
It is transferred in chromatographic column, the efflux Yu Shuizhong being washed till in chromatographic column with alcohol does not generate white opacity, is washed with deionized water
The alcohol of resin in chromatographic column, then processing is modified to macroporous weak base ion exchange resin with 1N hydrochloric acid solution, it is modified
Chromatographic column.
Further: in the step S10, the concentration of the ethyl alcohol is 40-50%, the concentration of the sodium-chloride water solution
For 20-30%.
Superoxide dismutase abbreviation SOD is a kind of biological enzyme for being widely present in nature, not by contained metal species
It is same to be divided into copper zinc SOD, manganese SOD and tri- kinds of iron SOD.
SOD can be catalyzed the reaction for removing ultra-oxygen anion free radical.Free radical be have unpaired valence electron atom or
Atomic group, molecule or ion are constituted.Under normal physiological condition, free radical, the generation of free radical are constantly be generated in organism
Equilibrium state is in removing.It, will be to DNA, protein and lipid when free radical yield is more but under certain pathologic conditions
Equal large biological molecules cause to damage, and lead to the generation of organism disease.It is that human body is raw since free radical has the chemical activity of height
The mesostate of a variety of biochemical reactions in life activity, free radical attack large biological molecule cause tissue damage to be many diseases
The root of occurrence and development.Thus SOD is damaged in defence organism from ultra-oxygen anion free radical, anti-radiation, antitumor and prolong
Slow body aging etc. plays an important role.
SOD have the function of remove body metabolic process in generates excessive ultra-oxygen anion free radical, can delay by
In the aging phenomenon that free radical is encroached on and is occurred, the i.e. appearance of delay skin aging and lipofuscin precipitating.Aging free radical theory
Think that aging is to be attached to destructive exercising result, free radical at random from generated free radical in body normal metabolic processes
Cause the main mechanism of body aging to can be summarized as following three aspect: (1) reducing the cross-linked polymeric and lipofuscin of large biological molecule
Accumulation；(2) slow down the histiocytic damage of organ and reduce；(3) reduction of immunocompetence is prevented.
The resistance radical damage that human body induces an illness to radical damage can be improved and supporting for inducing an illness in SOD
Drag mainly includes tumour, inflammation, pulmonary emphysema, cataract and autoimmune disease etc. when SOD adds as functional food ingredient
When entering in food, generation, the development of many diseases can be effectively suppressed, have very big effect to human health.
SOD can also remove organism fatigue, enhance the effect to the adaptive faculty of excess load large amount of exercise.In the big fortune of excess load
During momentum, portion of tissue cell can be alternately present transient ischemic and weight perfusion phenomenon in body, fill again after causing ischemic
Stream damage, the in addition increase of lactic acid production lead to the fatigue of muscle and damage.If supplying SOD before movement, muscle can be protected to keep away
Exempt from above-mentioned phenomenon occur.Single strong movements can make interior free yl horizontal significantly raised, and excessive free radical is as initiator
Unsaturated fatty acid in attack cells film causes lipid peroxidation, and membrane fluidity is caused to decline, brittleness enhancing.Lipid peroxide
A feature be generate malonaldehyde, formed phosphatide between crosslinking, can also with hemoglobin formed tan product.Body is through play
After strong movement, the ATP content creatine kinase vigor that decreases temporarily declines, and AMP content increases, in the effect of adenine trans-aminase
Under promote IMP to increase, then under the action of core seeks sour enzyme and core general enzyme, generate time xanthosine, after movement excessive time it is yellow he
Cry of certain animals forms excessive O under the action of secondary xanthosine oxidizing ferment2-.Therefore, supplemented with exogenous SOD can effectively inhibit movement to cause
Cellular damage.
Compared with prior art, the beneficial effects of the present invention are:
A kind of marine organisms source superoxide dismutase extraction and separation process of the invention, internal organ and fish blood system are not carried out
After coarse extraction, then the extracting solution obtained to centrifugation purifies, and extracts the superoxide dismutase of preparation, and purity is high, yield are high,
Its unit enzyme activity can achieve 6000U/mg or more.
A kind of marine organisms source superoxide dismutase extraction and separation process of the invention is carrying out internal organ coarse extraction and fish
The use that a large amount of organic solvent and inorganic salts sulphur ammonium in traditional extraction process are changed when blood coarse extraction, not only reduces because adding
Add the influence caused by these reagents to superoxide dismutase activity and safety, and reduce waste for reagent and
Subsequent removal technique reduces its industry cost.
A kind of marine organisms source superoxide dismutase extraction and separation process of the invention makes in carrying out purification process
Superoxide dismutase is protected with protective agent, to avoid heat, acid, destruction of the alkali to superoxide dismutase, is had
The activity for improving finished product superoxide dismutase of effect.
A kind of marine organisms source superoxide dismutase extraction and separation process of the invention, using macropore weak base ion exchange
Resin chromatography method purifies superoxide dismutase crude extract effectively, using macroreticular resin chromatographic technique to crude extract
It further isolates and purifies, the simple process, easily operated, purifying products are at low cost, is easily industrialized production.
A kind of marine organisms source superoxide dismutase extraction and separation process of the invention, raw material be ocean fish internal organ and
Fish blood effectively reduces because processing fishery -ies product generates environmental pollution and the wasting of resources brought by a large amount of by-products, realizes waste
It utilizes, turns waste into wealth.
Specific embodiment
In order to deepen the understanding of the present invention, the present invention will be described in further detail with reference to the examples below, implements below
Example for explaining only the invention, is not intended to limit the scope of the present invention..
Embodiment 1
A kind of marine organisms source superoxide dismutase extraction and separation process, specifically includes the following steps:
S1, it takes fresh ocean fish, takes out internal organ and fish blood, internal organ are cleaned up, drain rear spare, be added into fish blood
The sodium citrate of 5-10% fish blood quality, refrigerates spare after mixing；
S2, the internal organ cleaned up are beaten, obtain internal organ slurries；
S3, phosphate buffer solution is added into internal organ slurries, adjusts extracting solution pH6.5-7.5, stirs and extract at 40-60 DEG C
2-6h obtains internal organ extracting solution；
In the step S3, the additional amount of phosphate buffer solution is that 6-10L is added in every kilogram of internal organ slurries.
S4, heating removal foreign protein is carried out to internal organ extracting solution, rear centrifugation removal precipitating obtains supernatant A；
In the step S4, extracting solution is 60-80 DEG C by heating removal part foreign protein, heating temperature, heating time
For 60-140min.
S5, fish blood is placed in a centrifuge to centrifugation, red blood cell is made in sediment brine, broken with ultrasonic wave
Wall processing is added deionized water, obtains hemolysate after standing under ice bath；
In the step S5, the ultrasonic power of supersonic wave wall breaking processing is 150-250W, ultrasonic time 10-25min.
S6, the ethanol solution that 0-4 DEG C is added into hemolysate, add 0-4 DEG C of chloroform, then stir, stand, centrifugation
Supernatant is collected afterwards and obtains crude enzyme liquid, acetone is added into crude enzyme liquid, is then stirred, is centrifuged, taking precipitate；
In the step S6, the additional amount of ethanol solution is the volume of 20-40% hemolysate, and the additional amount of chloroform is 15-
The volume of 25% hemolysate, the additional amount of acetone are 3-4 times of volume of crude enzyme liquid.
S7, the obtained sediment of step S6 is dissolved with phosphate buffer, stir, be centrifuged after obtain supernatant, pass through
Dialysis removes salt ion, obtains supernatant C；
In the step S7, the revolving speed of centrifuge is 3000-5000r/min, centrifugation time 20-30min.
S8, merge supernatant A and supernatant C, obtain mixed liquor, mixed liquor is concentrated by ultrafiltration, obtain concentrate, it will
Concentrate heat temperature raising, adjusting pH value are 8.5-9.0, and add protective agent heat preservation, cooling after heat preservation, and centrifugation removal precipitating obtains
Supernatant D；
In the step S8, protective agent is lauroyl chloride, and the additive amount of lauroyl chloride is the 0.02- of concentrate quality
0.04%；Soaking time is 2-3h, is cooled to 10-20 DEG C after heat preservation, and supernatant is collected by centrifugation, and wherein centrifugal speed is 5000-
8000r/min。
S9, supernatant D is dissolved in the deionized water containing ethyl alcohol, the thick solution of SOD is obtained, with the stream of 30-70ml/h
Speed makes the thick solution of SOD pass through modified chromatographic column, then the remaining thick solution of SOD in modified chromatographic column is washed with deionized water；
In the step S9, the volume ratio of supernatant D and deionized water is 1:8-10, and the concentration of alcohol in deionized water is
20%.
In the step S9, modified chromatographic column the preparation method comprises the following steps: take macroporous weak base ion exchange resin, be transferred to chromatography
In column, the efflux Yu Shuizhong being washed till in chromatographic column with alcohol does not generate white opacity, is washed with deionized water tree in chromatographic column
The alcohol of rouge, then processing is modified to macroporous weak base ion exchange resin with 1N hydrochloric acid solution, obtain modified chromatographic column.
S10, the thick solution of SOD adsorbed with ethyl alcohol and sodium-chloride water solution elution macroporous weak base ion exchange resin, are collected
Efflux is concentrated, is freeze-dried up to the superoxide dismutase purified.
In the step S10, the concentration of the ethyl alcohol is 40-50%, and the concentration of the sodium-chloride water solution is 20-
30%.
Embodiment 2
A kind of marine organisms source superoxide dismutase extraction and separation process, specifically includes the following steps:
S1, it takes fresh ocean fish, takes out internal organ and fish blood, internal organ are cleaned up, drain rear spare, be added into fish blood
The sodium citrate of 5-10% fish blood quality, refrigerates spare after mixing；
S2, the internal organ cleaned up are beaten, obtain internal organ slurries；
S3, phosphate buffer solution is added into internal organ slurries, adjusts extracting solution pH6.5-7.5, stirs and extract at 40-60 DEG C
2-6h obtains internal organ extracting solution；
In the step S3, the additional amount of phosphate buffer solution is that 6-10L is added in every kilogram of internal organ slurries.
S4, heating removal foreign protein is carried out to internal organ extracting solution, rear centrifugation removal precipitating obtains supernatant A；
In the step S4, extracting solution is 60-80 DEG C by heating removal part foreign protein, heating temperature, heating time
For 60-140min.
S5, fish blood is placed in a centrifuge to centrifugation, red blood cell is made in sediment brine, broken with ultrasonic wave
Wall processing is added deionized water, obtains hemolysate after standing under ice bath；
In the step S5, the ultrasonic power of supersonic wave wall breaking processing is 150-250W, ultrasonic time 10-25min.
S6, the ethanol solution that 0-4 DEG C is added into hemolysate, add 0-4 DEG C of chloroform, then stir, stand, centrifugation
Supernatant is collected afterwards and obtains crude enzyme liquid, acetone is added into crude enzyme liquid, is then stirred, is centrifuged, taking precipitate；
In the step S6, the additional amount of ethanol solution is the volume of 20-40% hemolysate, and the additional amount of chloroform is 15-
The volume of 25% hemolysate, the additional amount of acetone are 3-4 times of volume of crude enzyme liquid.
S7, the obtained sediment of step S6 is dissolved with phosphate buffer, stir, be centrifuged after obtain supernatant, pass through
Dialysis removes salt ion, obtains supernatant C；
In the step S7, the revolving speed of centrifuge is 3000-5000r/min, centrifugation time 20-30min.
S8, merge supernatant A and supernatant C, obtain mixed liquor, mixed liquor is concentrated by ultrafiltration, obtain concentrate, it will
Concentrate heat temperature raising, adjusting pH value are 8.5-9.0, and add protective agent heat preservation, cooling after heat preservation, and centrifugation removal precipitating obtains
Supernatant D；
In the step S8, protective agent is lauroyl chloride, and the additive amount of lauroyl chloride is the 0.02- of concentrate quality
0.04%；Soaking time is 2-3h, is cooled to 10-20 DEG C after heat preservation, and supernatant is collected by centrifugation, and wherein centrifugal speed is 5000-
8000r/min。
S9, supernatant D is dissolved in the deionized water containing ethyl alcohol, the thick solution of SOD is obtained, with the stream of 30-70ml/h
Speed makes the thick solution of SOD pass through modified chromatographic column, then the remaining thick solution of SOD in modified chromatographic column is washed with deionized water；
In the step S9, the volume ratio of supernatant D and deionized water is 1:8-10, and the concentration of alcohol in deionized water is
20%.
In the step S9, modified chromatographic column the preparation method comprises the following steps: take macroporous weak base ion exchange resin, be transferred to chromatography
In column, the efflux Yu Shuizhong being washed till in chromatographic column with alcohol does not generate white opacity, is washed with deionized water tree in chromatographic column
The alcohol of rouge, then processing is modified to macroporous weak base ion exchange resin with 1N hydrochloric acid solution, obtain modified chromatographic column.
S10, the thick solution of SOD adsorbed with ethyl alcohol and sodium-chloride water solution elution macroporous weak base ion exchange resin, are collected
Efflux is concentrated, is freeze-dried up to the superoxide dismutase purified.
In the step S10, the concentration of the ethyl alcohol is 40-50%, and the concentration of the sodium-chloride water solution is 20-
30%.
Embodiment 3
A kind of marine organisms source superoxide dismutase extraction and separation process, specifically includes the following steps:
S1, it takes fresh ocean fish, takes out internal organ and fish blood, internal organ are cleaned up, drain rear spare, be added into fish blood
The sodium citrate of 5-10% fish blood quality, refrigerates spare after mixing；
S2, the internal organ cleaned up are beaten, obtain internal organ slurries；
S3, phosphate buffer solution is added into internal organ slurries, adjusts extracting solution pH6.5-7.5, stirs and extract at 40-60 DEG C
2-6h obtains internal organ extracting solution；
In the step S3, the additional amount of phosphate buffer solution is that 6-10L is added in every kilogram of internal organ slurries.
S4, heating removal foreign protein is carried out to internal organ extracting solution, rear centrifugation removal precipitating obtains supernatant A；
In the step S4, extracting solution is 60-80 DEG C by heating removal part foreign protein, heating temperature, heating time
For 60-140min.
S5, fish blood is placed in a centrifuge to centrifugation, red blood cell is made in sediment brine, broken with ultrasonic wave
Wall processing is added deionized water, obtains hemolysate after standing under ice bath；
In the step S5, the ultrasonic power of supersonic wave wall breaking processing is 150-250W, ultrasonic time 10-25min.
S6, the ethanol solution that 0-4 DEG C is added into hemolysate, add 0-4 DEG C of chloroform, then stir, stand, centrifugation
Supernatant is collected afterwards and obtains crude enzyme liquid, acetone is added into crude enzyme liquid, is then stirred, is centrifuged, taking precipitate；
In the step S6, the additional amount of ethanol solution is the volume of 20-40% hemolysate, and the additional amount of chloroform is 15-
The volume of 25% hemolysate, the additional amount of acetone are 3-4 times of volume of crude enzyme liquid.
S7, the obtained sediment of step S6 is dissolved with phosphate buffer, stir, be centrifuged after obtain supernatant, pass through
Dialysis removes salt ion, obtains supernatant C；
In the step S7, the revolving speed of centrifuge is 3000-5000r/min, centrifugation time 20-30min.
S8, merge supernatant A and supernatant C, obtain mixed liquor, mixed liquor is concentrated by ultrafiltration, obtain concentrate, it will
Concentrate heat temperature raising, adjusting pH value are 8.5-9.0, and add protective agent heat preservation, cooling after heat preservation, and centrifugation removal precipitating obtains
Supernatant D；
In the step S8, protective agent is lauroyl chloride, and the additive amount of lauroyl chloride is the 0.02- of concentrate quality
0.04%；Soaking time is 2-3h, is cooled to 10-20 DEG C after heat preservation, and supernatant is collected by centrifugation, and wherein centrifugal speed is 5000-
8000r/min。
S9, supernatant D is dissolved in the deionized water containing ethyl alcohol, the thick solution of SOD is obtained, with the stream of 30-70ml/h
Speed makes the thick solution of SOD pass through modified chromatographic column, then the remaining thick solution of SOD in modified chromatographic column is washed with deionized water；
In the step S9, the volume ratio of supernatant D and deionized water is 1:8-10, and the concentration of alcohol in deionized water is
20%.
In the step S9, modified chromatographic column the preparation method comprises the following steps: take macroporous weak base ion exchange resin, be transferred to chromatography
In column, the efflux Yu Shuizhong being washed till in chromatographic column with alcohol does not generate white opacity, is washed with deionized water tree in chromatographic column
The alcohol of rouge, then processing is modified to macroporous weak base ion exchange resin with 1N hydrochloric acid solution, obtain modified chromatographic column.
S10, the thick solution of SOD adsorbed with ethyl alcohol and sodium-chloride water solution elution macroporous weak base ion exchange resin, are collected
Efflux is concentrated, is freeze-dried up to the superoxide dismutase purified.
In the step S10, the concentration of the ethyl alcohol is 40-50%, and the concentration of the sodium-chloride water solution is 20-
30%.
Embodiment 4
A kind of marine organisms source superoxide dismutase extraction and separation process, specifically includes the following steps:
S1, it takes fresh ocean fish, takes out internal organ and fish blood, internal organ are cleaned up, drain rear spare, be added into fish blood
The sodium citrate of 5-10% fish blood quality, refrigerates spare after mixing；
S2, the internal organ cleaned up are beaten, obtain internal organ slurries；
S3, phosphate buffer solution is added into internal organ slurries, adjusts extracting solution pH6.5-7.5, stirs and extract at 40-60 DEG C
2-6h obtains internal organ extracting solution；
In the step S3, the additional amount of phosphate buffer solution is that 6-10L is added in every kilogram of internal organ slurries.
S4, heating removal foreign protein is carried out to internal organ extracting solution, rear centrifugation removal precipitating obtains supernatant A；
In the step S4, extracting solution is 60-80 DEG C by heating removal part foreign protein, heating temperature, heating time
For 60-140min.
S5, fish blood is placed in a centrifuge to centrifugation, red blood cell is made in sediment brine, broken with ultrasonic wave
Wall processing is added deionized water, obtains hemolysate after standing under ice bath；
In the step S5, the ultrasonic power of supersonic wave wall breaking processing is 150-250W, ultrasonic time 10-25min.
S6, the ethanol solution that 0-4 DEG C is added into hemolysate, add 0-4 DEG C of chloroform, then stir, stand, centrifugation
Supernatant is collected afterwards and obtains crude enzyme liquid, acetone is added into crude enzyme liquid, is then stirred, is centrifuged, taking precipitate；
In the step S6, the additional amount of ethanol solution is the volume of 20-40% hemolysate, and the additional amount of chloroform is 15-
The volume of 25% hemolysate, the additional amount of acetone are 3-4 times of volume of crude enzyme liquid.
S7, the obtained sediment of step S6 is dissolved with phosphate buffer, stir, be centrifuged after obtain supernatant, pass through
Dialysis removes salt ion, obtains supernatant C；
In the step S7, the revolving speed of centrifuge is 3000-5000r/min, centrifugation time 20-30min.
S8, merge supernatant A and supernatant C, obtain mixed liquor, mixed liquor is concentrated by ultrafiltration, obtain concentrate, it will
Concentrate heat temperature raising, adjusting pH value are 8.5-9.0, and add protective agent heat preservation, cooling after heat preservation, and centrifugation removal precipitating obtains
Supernatant D；
In the step S8, protective agent is lauroyl chloride, and the additive amount of lauroyl chloride is the 0.02- of concentrate quality
0.04%；Soaking time is 2-3h, is cooled to 10-20 DEG C after heat preservation, and supernatant is collected by centrifugation, and wherein centrifugal speed is 5000-
8000r/min。
S9, supernatant D is dissolved in the deionized water containing ethyl alcohol, the thick solution of SOD is obtained, with the stream of 30-70ml/h
Speed makes the thick solution of SOD pass through modified chromatographic column, then the remaining thick solution of SOD in modified chromatographic column is washed with deionized water；
In the step S9, the volume ratio of supernatant D and deionized water is 1:8-10, and the concentration of alcohol in deionized water is
20%.
In the step S9, modified chromatographic column the preparation method comprises the following steps: take macroporous weak base ion exchange resin, be transferred to chromatography
In column, the efflux Yu Shuizhong being washed till in chromatographic column with alcohol does not generate white opacity, is washed with deionized water tree in chromatographic column
The alcohol of rouge, then processing is modified to macroporous weak base ion exchange resin with 1N hydrochloric acid solution, obtain modified chromatographic column.
S10, the thick solution of SOD adsorbed with ethyl alcohol and sodium-chloride water solution elution macroporous weak base ion exchange resin, are collected
Efflux is concentrated, is freeze-dried up to the superoxide dismutase purified.
In the step S10, the concentration of the ethyl alcohol is 40-50%, and the concentration of the sodium-chloride water solution is 20-
30%.
Embodiment 5
A kind of marine organisms source superoxide dismutase extraction and separation process, specifically includes the following steps:
S1, it takes fresh ocean fish, takes out internal organ and fish blood, internal organ are cleaned up, drain rear spare, be added into fish blood
The sodium citrate of 5-10% fish blood quality, refrigerates spare after mixing；
S2, the internal organ cleaned up are beaten, obtain internal organ slurries；
S3, phosphate buffer solution is added into internal organ slurries, adjusts extracting solution pH6.5-7.5, stirs and extract at 40-60 DEG C
2-6h obtains internal organ extracting solution；
In the step S3, the additional amount of phosphate buffer solution is that 6-10L is added in every kilogram of internal organ slurries.
S4, heating removal foreign protein is carried out to internal organ extracting solution, rear centrifugation removal precipitating obtains supernatant A；
In the step S4, extracting solution is 60-80 DEG C by heating removal part foreign protein, heating temperature, heating time
For 60-140min.
S5, fish blood is placed in a centrifuge to centrifugation, red blood cell is made in sediment brine, broken with ultrasonic wave
Wall processing is added deionized water, obtains hemolysate after standing under ice bath；
In the step S5, the ultrasonic power of supersonic wave wall breaking processing is 150-250W, ultrasonic time 10-25min.
S6, the ethanol solution that 0-4 DEG C is added into hemolysate, add 0-4 DEG C of chloroform, then stir, stand, centrifugation
Supernatant is collected afterwards and obtains crude enzyme liquid, acetone is added into crude enzyme liquid, is then stirred, is centrifuged, taking precipitate；
In the step S6, the additional amount of ethanol solution is the volume of 20-40% hemolysate, and the additional amount of chloroform is 15-
The volume of 25% hemolysate, the additional amount of acetone are 3-4 times of volume of crude enzyme liquid.
S7, the obtained sediment of step S6 is dissolved with phosphate buffer, stir, be centrifuged after obtain supernatant, pass through
Dialysis removes salt ion, obtains supernatant C；
In the step S7, the revolving speed of centrifuge is 3000-5000r/min, centrifugation time 20-30min.
S8, merge supernatant A and supernatant C, obtain mixed liquor, mixed liquor is concentrated by ultrafiltration, obtain concentrate, it will
Concentrate heat temperature raising, adjusting pH value are 8.5-9.0, and add protective agent heat preservation, cooling after heat preservation, and centrifugation removal precipitating obtains
Supernatant D；
In the step S8, protective agent is lauroyl chloride, and the additive amount of lauroyl chloride is the 0.02- of concentrate quality
0.04%；Soaking time is 2-3h, is cooled to 10-20 DEG C after heat preservation, and supernatant is collected by centrifugation, and wherein centrifugal speed is 5000-
8000r/min。
S9, supernatant D is dissolved in the deionized water containing ethyl alcohol, the thick solution of SOD is obtained, with the stream of 30-70ml/h
Speed makes the thick solution of SOD pass through modified chromatographic column, then the remaining thick solution of SOD in modified chromatographic column is washed with deionized water；
In the step S9, the volume ratio of supernatant D and deionized water is 1:8-10, and the concentration of alcohol in deionized water is
20%.
In the step S9, modified chromatographic column the preparation method comprises the following steps: take macroporous weak base ion exchange resin, be transferred to chromatography
In column, the efflux Yu Shuizhong being washed till in chromatographic column with alcohol does not generate white opacity, is washed with deionized water tree in chromatographic column
The alcohol of rouge, then processing is modified to macroporous weak base ion exchange resin with 1N hydrochloric acid solution, obtain modified chromatographic column.
S10, the thick solution of SOD adsorbed with ethyl alcohol and sodium-chloride water solution elution macroporous weak base ion exchange resin, are collected
Efflux is concentrated, is freeze-dried up to the superoxide dismutase purified.
In the step S10, the concentration of the ethyl alcohol is 40-50%, and the concentration of the sodium-chloride water solution is 20-
30%.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and what is described in the above embodiment and the description is only the present invention
Principle, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these variation and
Improvement is both fallen in the range of claimed invention.The present invention claims protection scope by appended claims and its
Equivalent defines.
Claims (9)
1. a kind of marine organisms source superoxide dismutase extraction and separation process, which is characterized in that specifically includes the following steps:
S1, it takes fresh ocean fish, takes out internal organ and fish blood, internal organ are cleaned up, drain rear spare, 5- is added into fish blood
The sodium citrate of 10% fish blood quality, refrigerates spare after mixing；
S2, the internal organ cleaned up are beaten, obtain internal organ slurries；
S3, phosphate buffer solution is added into internal organ slurries, adjusts extracting solution pH6.5-7.5,2- is extracted in stirring at 40-60 DEG C
6h obtains internal organ extracting solution；
S4, heating removal foreign protein is carried out to internal organ extracting solution, rear centrifugation removal precipitating obtains supernatant A；
S5, fish blood is placed in a centrifuge to centrifugation, red blood cell is made, at supersonic wave wall breaking in sediment brine
Reason is added deionized water, obtains hemolysate after standing under ice bath；
S6, the ethanol solution that 0-4 DEG C is added into hemolysate, add 0-4 DEG C of chloroform, then stir, stand, receive after centrifugation
Collect supernatant and obtain crude enzyme liquid, acetone is added into crude enzyme liquid, then stirs, is centrifuged, taking precipitate；
S7, the obtained sediment of step S6 is dissolved with phosphate buffer, stir, be centrifuged after obtain supernatant, pass through dialysis
Salt ion is removed, supernatant C is obtained；
S8, merge supernatant A and supernatant C, obtain mixed liquor, mixed liquor is concentrated by ultrafiltration, obtain concentrate, will be concentrated
Liquid heat temperature raising, adjusting pH value are 8.5-9.0, and add protective agent heat preservation, cooling after heat preservation, and centrifugation removal precipitating obtains supernatant
Liquid D；
S9, supernatant D is dissolved in the deionized water containing ethyl alcohol, obtains the thick solution of SOD, is made with the flow velocity of 30-70ml/h
The thick solution of SOD passes through modified chromatographic column, then the remaining thick solution of SOD in modified chromatographic column is washed with deionized water；
S10, the thick solution of SOD adsorbed with ethyl alcohol and sodium-chloride water solution elution macroporous weak base ion exchange resin, collect outflow
Liquid is concentrated, is freeze-dried up to the superoxide dismutase purified.
2. a kind of marine organisms source superoxide dismutase extraction and separation process according to claim 1, which is characterized in that
In the step S3, the additional amount of phosphate buffer solution is that 6-10L is added in every kilogram of internal organ slurries.
3. a kind of marine organisms source superoxide dismutase extraction and separation process according to claim 1, which is characterized in that
In the step S4, extracting solution is 60-80 DEG C by heating removal part foreign protein, heating temperature, heating time 60-
140min。
4. a kind of marine organisms source superoxide dismutase extraction and separation process according to claim 1, which is characterized in that
In the step S5, the ultrasonic power of supersonic wave wall breaking processing is 150-250W, ultrasonic time 10-25min.
5. a kind of marine organisms source superoxide dismutase extraction and separation process according to claim 1, which is characterized in that
In the step S6, the additional amount of ethanol solution is the volume of 20-40% hemolysate, and the additional amount of chloroform is 15-25% haemolysis
The volume of liquid, the additional amount of acetone are 3-4 times of volume of crude enzyme liquid.
6. a kind of marine organisms source superoxide dismutase extraction and separation process according to claim 1, which is characterized in that
In the step S7, the revolving speed of centrifuge is 3000-5000r/min, centrifugation time 20-30min.
7. a kind of marine organisms source superoxide dismutase extraction and separation process according to claim 1, which is characterized in that
In the step S8, protective agent is lauroyl chloride, and the additive amount of lauroyl chloride is the 0.02-0.04% of concentrate quality；Heat preservation
Time is 2-3h, is cooled to 10-20 DEG C after heat preservation, and supernatant is collected by centrifugation, and wherein centrifugal speed is 5000-8000r/min.
8. a kind of marine organisms source superoxide dismutase extraction and separation process according to claim 1, which is characterized in that
In the step S9, modified chromatographic column the preparation method comprises the following steps: take macroporous weak base ion exchange resin, be transferred in chromatographic column, use
The efflux Yu Shuizhong that alcohol is washed till in chromatographic column does not generate white opacity, is washed with deionized water the wine of resin in chromatographic column
Essence, then processing is modified to macroporous weak base ion exchange resin with 1N hydrochloric acid solution, obtain modified chromatographic column.
9. a kind of marine organisms source superoxide dismutase extraction and separation process according to claim 1, which is characterized in that
In the step S10, the concentration of the ethyl alcohol is 40-50%, and the concentration of the sodium-chloride water solution is 20-30%.
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