CN107083380A - A kind of low-temperature superoxide dismutase protective agent and preparation method thereof - Google Patents
A kind of low-temperature superoxide dismutase protective agent and preparation method thereof Download PDFInfo
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- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
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Abstract
The present invention relates to a kind of low-temperature superoxide dismutase protective agent and preparation method thereof.The protective agent is composed of the following components：Low-temperature superoxide dismutase, polyethylene glycol, dithiothreitol (DTT) (DTT), Sodium Hyaluronate, imidazolidinyl urea, trehalose, deionized water.Its preparation method is：Trehalose, polyethylene glycol are added to be sterilized in deionized water, obtains base fluid；Sodium Hyaluronate and DTT are added into low-temperature superoxide dismutase, stir filtration sterilization, obtains low-temperature superoxide dismutase mixed solution；Low-temperature superoxide dismutase mixed solution is added into base fluid under aseptic condition, imidazolidinyl urea is then added, is mixed evenly, plus deionized water adjusts to obtain described superoxide dismutase protective agent.The low-temperature superoxide dismutase protective agent of the present invention makes the enzyme activity of superoxide dismutase keep stable, and cost of manufacture is cheap, can greatly reduce enzyme activity loss, improve enzyme service life and application effect.
Description
Technical field
The present invention relates to bioengineering field, and in particular to a kind of low-temperature superoxide dismutase protective agent and its preparation side
Method.
Background technology
Superoxide dismutase (l.15.1.1 superoxide dismutase, EC, write a Chinese character in simplified form SOD) is that a class is deposited extensively
It is the metalloenzyme in organism, is catalysis ultra-oxygen anion free radical (O2 -) and hydrogen ion generation hydrogen peroxide and molecular oxygen,
So as to remove the oxygen radical in organism, play and protect organisms from injury effect.By the kind quasi-SOD of institute's metal ion
It is divided into four classes：One class is blue-green copper-zinc superoxide dismutase (Cu/Zn-SOD), be primarily present in eukaryotic cytosol and
It is most study, a most deep class in chloroplaset；Equations of The Second Kind is that aubergine nickel superoxide dismutase (Ni-SOD) and manganese are super
Superoxide dismutase (Mn-SOD), is primarily present in prokaryote and mitochondrial matrix；3rd class is yellowish-brown iron
Superoxide dismutase (Fe-SOD), is primarily present in prokaryotic and some plants.
SOD is medically mainly used in adjuvant radiotherapy and chemotherapy as biological enzyme formulation, the protection of the organ such as kidney, liver, heart
And transplanting, eliminate radiation-induced side effect, and the probe as some diseases etc.；Food industry as additive application in
Beverage and beer etc..In recent years SOD be widely used in treatment oxygen poisoning, it is cataract of old people, diabetes, angiocardiopathy, each
Plant a variety of diseases such as inflammation；SOD is always domestic and international study hotspot in decades, initialization phase focus mostly in from animal blood or
Extracted in plant tissue and SOD complex process is extracted in SOD, but animal and plant body, security is low, and micro-organisms SOD has life
The production cycle is short, zymotechnique is simple, production efficiency is high, nontoxic and high pick-up rate the features such as receive significant attention.Domestic and foreign scholars
It is main to research and produce the side such as seed selection, fermentation technology optimization, the clonal expression of gene and product development of high temperature SOD microorganisms
Face, and low temperature SOD optimum temperatures are generally 35~40 DEG C, the relatively physiological temp of human body, applied to therapeutic effect
It is obvious.Low temperature SOD is extremely sensitive to thermal response, and with high enzymatic activity and high catalytic efficiency at a temperature of natural environment,
It can lose the vigor of cold-adapted enzyme by gentle heat treatment, if SOD is applied to food industry to greatly shorten place
The time of reason process and save costliness expense is heated or cooled, and do not influence product quality, this will be helpful to pushing away for low temperature SOD
It is wide and use, fundamentally break away from cumbersome extraction process and heating, cooling device and the flow of middle temperature enzyme.In view of low temperature SOD
Application advantage under nature and physiological temp, marine low temperature SOD has extensive in terms of health care, food, cosmetics
Application prospect and potentiality to be exploited.
In current superoxide dismutase production, drying process is important means.Because superoxide dismutase to it is thermo-responsive,
Easily oxidized etc. good, most of factory, which selects, to be freeze-dried as drying process, but freeze drying process high energy consumption, investment
High, efficiency is low, production cost is high；And freezing dry process can lose about more than 10% SOD enzyme activity, and add when lyophilized
The additive entered, seriously governs the commercial application of superoxide dismutase industry.In consideration of it, providing a kind of new super oxygen
Compound is disproportionated enzymatic protective reagent, and stable enzymatic activity overcomes technical problem present in prior art, can keep superoxide dismutase
Stability in the stability of enzyme in process of production, the especially technique such as freeze-drying, improves the enzyme activity yield of producing enzyme, is this
Art personnel technical problem urgently to be resolved hurrily.
The content of the invention
In order to solve the above mentioned problem of prior art presence, the present invention is provided a kind of simple to operate, with low cost and can had
Preparation of effect protection low-temperature superoxide dismutase activity and preparation method thereof.
The technical solution adopted in the present invention is：
A kind of low-temperature superoxide dismutase protective agent：Raw material including following weight parts, low-temperature superoxide dismutase
300-500 parts, 100-300 parts of polyethylene glycol, 11-54 parts of Sodium Hyaluronate, 2-6 parts of dithiothreitol (DTT) (DTT), imidazolidinyl
1-3 parts of urea, 5-15 parts of trehalose, 122-582 parts of deionized water.
Further, described low-temperature superoxide dismutase protective agent, includes the raw material of following weight parts, low temperature super oxygen
400 parts of compound mutase, polyethylene glycol 200 part, 28 parts of Sodium Hyaluronate, 4 parts of DTT, 2 parts of imidazolidinyl urea, trehalose 10
Part, 356 parts of deionized water.
The preparation method of described low-temperature superoxide dismutase is：
(1) preparation of crude enzyme liquid：Sterile working, sticks Pseudoalteromonas by the sea activated and is inoculated into liquid fermentation and culture
In base, in 20 DEG C, 160r/min fermentation 48h, zymocyte liquid is in 4 DEG C, 6 000r/min centrifugation 30min, obtains wet thallus, 4 DEG C,
6 000r/min centrifuge 30min, and wet thallus phosphate buffer dissolves, (broken in crushing 30min under 480W ultrasonic wave ice baths
5s, is spaced 5s), after crushing, 4 DEG C, 12 000r/min centrifugation 15min, supernatant is crude enzyme liquid, aforesaid liquid fermentation medium
Being formulated percentage is：Glucose 2.0, yeast extract 0.5, Chen Haishui is prepared, and pH is natural；
(2) ammonium sulfate precipitation：6 parts of 25ml crude enzyme liquids are prepared, the cold ammonium sulfate of solid is added thereto respectively, makes its saturation molten
Liquid concentration difference 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 4 DEG C of 16h that saltout, pH7.8 phosphate buffers
Protein precipitation is dissolved, the active highest ammonium sulfate precipitation condition of low-temperature superoxide dismutase is determined；
(3) it is concentrated by ultrafiltration：Completion sample of saltouing is transferred to (molecular cut off 10KDa) super filter tube by pretreatment, 4
DEG C, 6000r/min, ultrafiltration 30min remove SO42-With part small molecular protein, liquid-transfering gun carefully takes out concentrate, 4 DEG C, protected
Hide standby, be partially soluble in phosphate buffer, determine low-temperature superoxide dismutase enzyme activity, calculate protein recovery；
(4) Sephadex G-100 column chromatographies：Sephadex G-100 gel columns are balanced with pH7.8 phosphate buffers, are treated
After baseline balance, enzyme liquid after 2ml ultrafiltration is taken to add the equilibrated Sephadex G-100 gel columns (Ф of pH7.8 phosphate buffers
1.6 cm × 60cm), regulation sets automatic collector, and often pipe collects 2ml or so, according to the eluting peak on display, it is determined that containing
Low-temperature superoxide dismutase enzyme is respectively managed, and is collected and is determined its enzyme activity, and merging is respectively managed containing enzyme activity, and 4 DEG C, preservation is standby；
(5) it is spray-dried：It is 60 DEG C, pressure position that the parameter of spray drying, which is set to 110 DEG C of inlet temperature, outlet temperature,
2kgf/cm2, flow rate of liquid be 5mL/min, trehalose, sucrose, the consumption of sodium alginate are in the protective agent in spray drying
2%-5%, the consumption of soluble starch is 6%-8%, and protectant total consumption control obtain low temperature super oxygen within 10%
Compound mutase.
The molecular weight polyethylene glycol is 4000-8000.
The protectant preparation method of low-temperature superoxide dismutase, comprises the following steps：
(1) trehalose, polyethylene glycol are added in deionized water, are stirred and are sterilized, and obtain base fluid, wherein trehalose,
Polyethylene glycol can direct high-temperature heat sterilization, do not interfere with function；
(2) Sodium Hyaluronate and DTT are added in low-temperature superoxide dismutase, and stirred, filtration sterilization must be low
Warm superoxide dismutase mixed solution, it is standby；, it is necessary to be examined to low-temperature superoxide dismutase activity after filtration sterilization
Survey, detection uses GB/T5009.171-2003 standards, and assay NBT photoreduction so can be to low temperature superoxide dismutase
The activity of enzyme has a quantification, facilitates the later stage to allocate；
(3) aseptically, low-temperature superoxide dismutase mixed solution is added into base fluid, then adds imidazolidinyl
Urea, is mixed evenly, and then adds deionized water regulation, obtains described superoxide dismutase protective agent, is added to base fluid low
Before warm superoxide dismutase mixed solution, base fluid is first cooled to 25-60 DEG C, high temperature low temperature superoxides discrimination is prevented
Change the activity of enzyme.
Further, in step (1), it is described sterilizing be moist heat sterilization, moist heat sterilization refer to saturated steam, boiling water or
The method that flowing steam is sterilized, using high-temperature high-pressure steam as medium, because steam potential is big, penetration power is strong, easily makes
Protein denaturation or solidification, ultimately result in the death of microorganism, so the sterilizing efficiency of the method is higher than hot-air sterilization, it is biological
The most frequently used sterilizing methods in the production process of field.
Further, the sterilising temp is 115-125 DEG C, and the sterilization time is 20-30min.
Further, filtration sterilization described in step (2) is to carry out filtration sterilization using bacteria filter.Typical filter device has thin
Film bacteria filter, ceramic bacteria filter, asbestos bacteria filter etc.；Filtration sterilization is using the method for physics detention by the bacterium of liquid or air
Remove, to reach sterile purpose.
Further, in step (3), deionized water need to be added after sterilizing.
Further, the deionized water is sterilized through moist hear heat test；The temperature of the sterilizing is 115-125 DEG C,
The sterilization time is 20-30min.
Superoxide dismutase (superoxide dismutase, EC l.15.1.1, SOD) is that one kind comes from life entity
Active material, the ultra-oxygen anion free radical that organism produces in metabolic processes can be removed.It is used herein
Low-temperature superoxide dismutase by being extracted in microorganism, it is fermented acquisition low-temperature superoxide dismutase crude enzyme liquid.SOD is only
Have under with bioactivity state, just the function with enzyme.That is " one is clear, five resist, one improves ", one is clear：Remove excess
Ultra-oxygen anion free radical；Five resist i.e.：Anti-oxidant, radioresistance, anti-aging, antifatigue, anti-inflammatory；One improves：Body is improved to exempt from
Epidemic disease power.
Sodium Hyaluronate is main to be made from Lactococcus fermentation, is white or off-white color particle or powder, odorless.Extensively
For cosmetic industry, it can keep that skin moisturizing is smooth, fine and smooth tender, high resilience, with wrinkle resistant, crease-resistant, beauty and health care etc.
Physiological action.And Sodium Hyaluronate has fresh-keeping and keep-alive effect to SOD.
Polyethylene glycol is nontoxic, nonirritant, mildly bitter flavor, with good water solubility, with moisture retention, lubricity, is changing
The sector applications such as cosmetic, pharmacy are more.
Imidazolidinyl urea is white powder, is used in cosmetics frequently as preservative.
Trehalose is also known as Radix Rhapontici seu Radix Echinopsis sugar, gill fungus sugar etc., is a kind of safe and reliable natural carbohydrate.Trehalose has to organism
Magical protective effect, is because trehalose in high temperature, high and cold, hyperosmosis and is dried under the severe environmental conditions such as dehydration thin
Cellular surface can form the diaphragm of uniqueness, and effectively protected protein matter molecule consistency is inactivated, so that the life for the body that sustains life
Process and biological characteristic.Many adverse circumstances to external world show the species of outstanding degeneration-resistant tolerance, all exist in vivo with them
Substantial amounts of trehalose has direct relation.And such as other carbohydrates of sucrose, glucose in nature, do not possess this function.
This unique functional characteristic so that trehalose is except that can be used as the excellent of pharmaceutical grade protein, enzyme, vaccine and other biological product
Beyond good activity protecting agent, cytoactive, the important component of moisturizing class cosmetics are also to maintain, more can be as preventing food bad
The particular foodstuff dispensing change, keep food fresh flavor, lifted food quality.
The present invention beneficial outcomes be：The low temperature contained in low-temperature superoxide dismutase protective agent of the present invention surpasses
Superoxide dismutase is extracted from microorganism, and fermented acquisition low-temperature superoxide dismutase crude enzyme liquid, its activity is higher；It is transparent
Matter acid sodium has fresh-keeping effect to low-temperature superoxide dismutase；Polyethylene glycol is a kind of protective agent of bioactivity；Imidazoles
Ureine has antisepsis；Trehalose has certain protective effect to various organized enzymes.Low temperature i.e. of the present invention surpasses
Various raw materials, which are organically combined together, in superoxide dismutase protective agent effectively to protect low-temperature superoxide dismutase to live
Property, and cost of manufacture is relatively low.In addition, each raw material is cosmetics in low-temperature superoxide dismutase protective agent of the present invention
Adaptable raw material in production.And SOD applies more in cosmetic industry, but it extensively should because of the unstable definite limitation of enzymatic activity
With, and preparation of the present invention can be directly used for the raw material of Cosmetic Manufacture, and low-temperature superoxide dismutase enzyme can be kept
It is active steady in a long-term.
Embodiment
With reference to embodiment, the present invention is described in detail.
Embodiment 1
A kind of low-temperature superoxide dismutase protective agent, includes the raw material of following weight parts, low-temperature superoxide dismutase
300mL, polyethylene glycol 100g, Sodium Hyaluronate 11g, DTT 2g, imidazolidinyl urea 1g, trehalose 5g, deionized water 122g.
The preparation method of described low-temperature superoxide dismutase is：
(1) preparation of crude enzyme liquid：Sterile working, sticks Pseudoalteromonas by the sea activated and is inoculated into liquid fermentation and culture
In base, in 20 DEG C, 160r/min fermentation 48h, zymocyte liquid is in 4 DEG C, 6 000r/min centrifugation 30min, obtains wet thallus, 4 DEG C,
6 000r/min centrifuge 30min, and wet thallus phosphate buffer dissolves, (broken in crushing 30min under 480W ultrasonic wave ice baths
5s, is spaced 5s), after crushing, 4 DEG C, 12 000r/min centrifugation 15min, supernatant is crude enzyme liquid, aforesaid liquid fermentation medium
Being formulated percentage is：Glucose 2.0, yeast extract 0.5, Chen Haishui is prepared, and pH is natural；
(2) ammonium sulfate precipitation：6 parts of 25ml crude enzyme liquids are prepared, the cold ammonium sulfate of solid is added thereto respectively, makes its saturation molten
Liquid concentration difference 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 4 DEG C of 16h that saltout, pH7.8 phosphate buffers
Protein precipitation is dissolved, the active highest ammonium sulfate precipitation condition of low-temperature superoxide dismutase is determined；
(3) it is concentrated by ultrafiltration：Completion sample of saltouing is transferred to (molecular cut off 10KDa) super filter tube by pretreatment, 4
DEG C, 6000r/min, ultrafiltration 30min remove SO42-With part small molecular protein, liquid-transfering gun carefully takes out concentrate, 4 DEG C, protected
Hide standby, be partially soluble in phosphate buffer, determine low-temperature superoxide dismutase enzyme activity, calculate protein recovery；
(4) Sephadex G-100 column chromatographies：Sephadex G-100 gel columns are balanced with pH7.8 phosphate buffers, are treated
After baseline balance, enzyme liquid after 2ml ultrafiltration is taken to add the equilibrated Sephadex G-100 gel columns (Ф of pH7.8 phosphate buffers
1.6 cm × 60cm), regulation sets automatic collector, and often pipe collects 2ml or so, according to the eluting peak on display, it is determined that containing
Low-temperature superoxide dismutase enzyme is respectively managed, and is collected and is determined its enzyme activity, and merging is respectively managed containing enzyme activity, and 4 DEG C, preservation is standby；
(5) it is spray-dried：It is 60 DEG C, pressure position that the parameter of spray drying, which is set to 110 DEG C of inlet temperature, outlet temperature,
2kgf/cm2, flow rate of liquid be 5mL/min, trehalose, sucrose, the consumption of sodium alginate are in the protective agent in spray drying
2%-5%, the consumption of soluble starch is 6%-8%, and protectant total consumption control obtain low temperature super oxygen within 10%
Compound mutase.
The protectant preparation method of low-temperature superoxide dismutase, comprises the following steps：
(1) trehalose, polyethylene glycol are added in deionized water, and stir carry out high-temperature heat sterilization, temperature during sterilizing
Control is at 115 DEG C, and sterilize 20min at this temperature, obtains base fluid；
(2) Sodium Hyaluronate and DTT are added in low-temperature superoxide dismutase, and stirred, then use filter
It is standby to the low-temperature superoxide dismutase mixed solution filtration sterilization added with Sodium Hyaluronate and DTT；After filtration sterilization,
Need to detect low-temperature superoxide dismutase activity, detection uses GB/T5009.171-2003 standards, and pyrogallol is certainly
Oxidizing process, can so have a quantification to the activity of low-temperature superoxide dismutase, facilitate the later stage to allocate；
(3) aseptically, low-temperature superoxide dismutase mixed solution is added into base fluid, then adds imidazolidinyl
Urea, is mixed evenly, and then adds deionized water regulation, obtains described low-temperature superoxide dismutase protective agent.Deionized water
Before addition, sterilized using moist hear heat test, sterilising temp is controlled at 120 DEG C, at this temperature, sterilize 25min.
Embodiment 2
A kind of low-temperature superoxide dismutase protective agent, includes the raw material of following weight parts, low-temperature superoxide dismutase
400mL, polyethylene glycol 200 g, Sodium Hyaluronate 30g, DTT 4g, imidazolidinyl urea 2g, trehalose 10g, deionized water 312g.
The preparation method be the same as Example 1 of described low-temperature superoxide dismutase.
The protectant preparation method of low-temperature superoxide dismutase, comprises the following steps：
(1) trehalose, polyethylene glycol are added in deionized water, and stir carry out high-temperature heat sterilization, temperature during sterilizing
Control is at 120 DEG C, and sterilize 25min at this temperature, obtains base fluid；
(2) Sodium Hyaluronate and DTT are added in low-temperature superoxide dismutase, and stirred, then use filter
It is standby to the low-temperature superoxide dismutase mixed solution filtration sterilization added with Sodium Hyaluronate and DTT；
(3) aseptically, low-temperature superoxide dismutase mixed solution is added into base fluid, then adds imidazolidinyl
Urea, is mixed evenly, and then adds deionized water regulation, obtains described low-temperature superoxide dismutase protective agent, deionized water
Before addition, sterilized using moist hear heat test, temperature control during sterilizing is at 120 DEG C, and sterilize 25min at this temperature.
Embodiment 3
A kind of low-temperature superoxide dismutase protective agent, includes the raw material of following weight parts, low-temperature superoxide dismutase
500mL, Liquid Macrogol g, Sodium Hyaluronate 54g, DTT 6g, imidazolidinyl urea 3g, trehalose 15g, deionized water 582g.
The preparation method be the same as Example 1 of described low-temperature superoxide dismutase.
The protectant preparation method of low-temperature superoxide dismutase, comprises the following steps：
(1) trehalose, polyethylene glycol are added in deionized water, and stir carry out high-temperature heat sterilization, temperature during sterilizing
Control is at 125 DEG C, and sterilize 30min at this temperature, obtains base fluid；
(2) Sodium Hyaluronate and DTT are added in low-temperature superoxide dismutase, and stirred, then use filter
It is standby to the low-temperature superoxide dismutase mixed solution filtration sterilization added with Sodium Hyaluronate and DTT；
(3) aseptically, low-temperature superoxide dismutase mixed solution is added into base fluid, then adds imidazolidinyl
Urea, is mixed evenly, and then adds deionized water regulation, obtains described low-temperature superoxide dismutase protective agent.Deionized water
Before addition, sterilized using moist hear heat test, temperature control during sterilizing is at 125 DEG C, and sterilize 30min at this temperature.
Embodiment 4
The measure of low-temperature superoxide dismutase enzyme activity, control group is liquid cryogen superoxide dismutase.
Determine each bacterial strain crude enzyme liquid low-temperature superoxide dismutase enzyme activity respectively using assay NBT photoreduction.In test tube
It is middle to add 0.1mol/L Tris-HCl buffer solutions, distilled water, low-temperature superoxide dismutase enzyme liquid according to table 1 respectively and (or implement
Example 1,2 and 3 low temperature SOD protective agents), 10mmol/L HCl, in after 20 DEG C of constant temperature 20min, added in sample cell and control tube
20 DEG C of preheated pyrogallols (pyrogallol addition is on the basis of mouse thymus cells speed is 0.07/min), rapidly
Shake up, immediately moved into sample in quartz cuvette with liquid-transfering gun, determine a light absorption value per 30s at wavelength 325nm.Survey altogether
3min。
The assay NBT photoreduction of table 1 determines low temperature SOD sample-adding tables
V1：Reaction solution cumulative volume, mL；V2：Determination sample volume, mL；n：Sample diluting liquid multiple；
ODA：Mouse thymus cells speed；ODB：Sample OD325Value changes speed
Enzyme activity is defined：Suppress the enzyme amount of mouse thymus cells speed 50% so that 1 milliliter of reaction solution is per minute, be a super oxygen
Compound mutase enzyme activity unit.The definition of above enzyme activity is modified slightly herein, even benzene three is suppressed so that every gram of wet thallus is per minute
The enzyme amount of phenol autoxidation speed 50%, is a superoxide dismutase enzyme activity unit.
The enzyme activity of the low-temperature superoxide dismutase of the different storage times of table 1
As shown in Table 1, with the extension of standing time, the activity of each low-temperature superoxide dismutase is gradually reduced, and is not added
Enter the protectant control group fall of liquid enzymes and be significantly greater than the fall for adding protectant each embodiment.Standing time
For 12 months, the low-temperature superoxide dismutase enzyme activity of control group was 79.28U/mL, and enzyme activity storage rate is 37.33%, each to implement
The low-temperature superoxide dismutase enzyme activity of example is 154.29-200.16U/mL, and enzyme activity storage rate is up to 72.65-94.25%.By
This is visible, and low-temperature superoxide dismutase protective agent of the invention can substantially reduce the loss of low-temperature superoxide dismutase enzyme activity,
Greatly improve the service life and application effect of low-temperature superoxide dismutase.
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert
The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
Claims (10)
1. a kind of low-temperature superoxide dismutase protective agent, it is characterised in that：Raw material including following weight parts, low temperature super oxygen
300-500 parts of thing mutase, 100-300 parts of polyethylene glycol, 11-54 parts of Sodium Hyaluronate, 2-6 parts of dithiothreitol (DTT), imidazolidine
1-3 parts of base urea, 5-15 parts of trehalose, 122-582 parts of deionized water.
2. a kind of low-temperature superoxide dismutase protective agent according to claim 1, it is characterised in that：Including following weight
The raw material of part, 400 parts of superoxide dismutase, polyethylene glycol 200 part, 28 parts of Sodium Hyaluronate, 4 parts of dithiothreitol (DTT), imidazoles
2 parts of ureine, 10 parts of trehalose, 356 parts of deionized water.
3. a kind of low-temperature superoxide dismutase protective agent according to claim 1-2 any one, it is characterised in that：Institute
The preparation method for stating low-temperature superoxide dismutase is：
(1) preparation of crude enzyme liquid：Sterile working, sticks Pseudoalteromonas by the sea activated and is inoculated into liquid fermentation medium
In, in 20 DEG C, 160r/min fermentation 48h, zymocyte liquid is in 4 DEG C, and 6 000r/min centrifugation 30min obtain wet thallus, 4 DEG C, 6
000r/min centrifuges 30min, and wet thallus phosphate buffer dissolves.In crushed under 480W ultrasonic wave ice baths 30min (broken 5s,
It is spaced 5s), after crushing, 4 DEG C, 12 000r/min centrifugation 15min, supernatant is crude enzyme liquid, aforesaid liquid fermentative medium formula
Percentage is：Glucose 2.0, yeast extract 0.5, Chen Haishui is prepared, and pH is natural；
(2) ammonium sulfate precipitation：6 parts of 25ml crude enzyme liquids are prepared, the cold ammonium sulfate of solid is added thereto respectively, makes its saturated solution dense
Degree difference 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 4 DEG C of 16h that saltout, the dissolving of pH7.8 phosphate buffers
Protein precipitation, determines the active highest ammonium sulfate precipitation condition of low-temperature superoxide dismutase；
(3) it is concentrated by ultrafiltration：By saltout complete sample be transferred to through pretreatment molecular cut off 10KDa super filter tube, 4 DEG C,
6000r/min, ultrafiltration 30min, remove SO42-With part small molecular protein, liquid-transfering gun carefully takes out concentrate, and 4 DEG C, preservation is standby
With being partially soluble in phosphate buffer, determine low-temperature superoxide dismutase enzyme activity, calculate protein recovery；
(4) Sephadex G-100 column chromatographies：Sephadex G-100 gel columns are balanced with pH7.8 phosphate buffers, baseline is treated
After balance, enzyme liquid after 2ml ultrafiltration is taken to add equilibrated Sephadex G-100 gel columns (the Ф 1.6cm of pH7.8 phosphate buffers
× 60cm), regulation sets automatic collector, and often pipe collects 2ml or so, according to the eluting peak on display, it is determined that super containing low temperature
Superoxide dismutase is respectively managed, and is collected and is determined its enzyme activity, and merging is respectively managed containing enzyme activity, and 4 DEG C, preservation is standby；
(5) it is spray-dried：It is 60 DEG C, pressure position 2kgf/ that the parameter of spray drying, which is set to 110 DEG C of inlet temperature, outlet temperature,
cm2, flow rate of liquid be 5mL/min, trehalose, sucrose, the consumption of sodium alginate are 2%- in the protective agent in spray drying
5%, the consumption of soluble starch is 6%-8%, and protectant total consumption control obtain low temperature superoxides within 10%
Mutase.
4. a kind of low-temperature superoxide dismutase protective agent according to claim 1-2 any one, it is characterised in that：System
Preparation Method comprises the following steps：
(1) trehalose, polyethylene glycol are added in deionized water, are stirred and are sterilized, obtain base fluid；
(2) Sodium Hyaluronate and dithiothreitol (DTT) are added in superoxide dismutase, and stirred, filtration sterilization obtains super oxygen
Compound mutase mixed solution, it is standby；
(3) aseptically, low-temperature superoxide dismutase mixed solution is added into base fluid, then adds imidazolidinyl urea,
It is mixed evenly, then adds deionized water regulation, obtain described low-temperature superoxide dismutase protective agent.
5. a kind of low-temperature superoxide dismutase protective agent according to claim 1-2 any one, it is characterised in that institute
Molecular weight polyethylene glycol is stated for 4000-8000.
6. a kind of low-temperature superoxide dismutase protective agent according to claim 4, it is characterised in that：Step (1) is described
Sterilize as moist heat sterilization.
7. a kind of low-temperature superoxide dismutase protective agent according to claim 6, it is characterised in that：The sterilising temp
For 115-125 DEG C, the sterilization time is 20-30min.
8. a kind of low-temperature superoxide dismutase protective agent according to claim 4, it is characterised in that：In step (2), institute
It is to carry out filtration sterilization using bacteria filter to state filtration sterilization.
9. a kind of low-temperature superoxide dismutase protective agent according to claim 4, it is characterised in that：In step (3), go
Ionized water need to be added after sterilizing.
10. a kind of low-temperature superoxide dismutase protective agent according to claim 9, it is characterised in that：The deionization
Water is sterilized through moist hear heat test；The temperature of the sterilizing is 115-125 DEG C, and the sterilization time is 20-30min.
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