CN110760487B - Method for preparing superoxide dismutase enriched dry powder - Google Patents
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Abstract
The invention belongs to the technical field of biological waste recycling, and particularly relates to a method for preparing superoxide dismutase enriched dry powder.
Description
Technical Field
The invention belongs to the technical field of biological waste recycling, and particularly relates to a method for preparing superoxide dismutase enriched dry powder.
Background
Superoxide Dismutase (SOD) is a kind of metalloenzyme which is widely present in organisms and can prevent oxidative damage, and is a well-known efficient oxygen radical scavenger. Research shows that the oxygen free radical scavenging has a very positive effect on maintaining good living states of organisms. Therefore, the superoxide dismutase has higher application value and market unit price, and particularly has higher acceptance in the markets of medicines, health-care foods, cosmetics and the like.
So far, SOD has been isolated from various organisms including bacteria, fungi, algae, plants and animals, but extraction of SOD from animals has a potential safety problem and is prohibited from use by the European Union and the like. Therefore, the application of plant resources to prepare SOD is an exploitable way. The crude extraction method of SOD in the prior art mainly comprises the following steps: ultrasonic extraction, enzyme extraction, phosphate buffer extraction, Tris-HCl and thermal denaturation, wherein the crude extract obtained by the above method contains a large amount of hetero-proteins, and precipitation methods such as thermal denaturation, acetone fractional precipitation, ammonium sulfate salting-out, chromatography, etc. are commonly used for purifying SOD to increase the specific activity of enzyme by selectively precipitating impurities or selectively precipitating desired components.
The research on the extraction and purification process of superoxide dismutase from loquat seeds (Wanglijun, loquat seed superoxide dismutase research [ J ]. food industry, 2015(3): 173-: the optimal technological conditions for extracting superoxide dismutase from loquat seeds are as follows: the ultrasonic power is 295.4W, the extraction time is 22.8min, the pH value is 6.7, and the liquid-material ratio is 20.8: 1 (mL/g). The separation and purification process of superoxide dismutase from loquat seeds comprises the following steps: sequentially carrying out thermal denaturation treatment, acetone precipitation and G-75 sephadex gel filtration to finally obtain the loquat seed superoxide dismutase with the protein removal rate of 92.1 percent, the total enzyme activity of 1091.6U, the enzyme specific activity of 4746.1U/mg and the purification multiple of 56.5. For another example, chinese patent document CN101629167A discloses a method for extracting superoxide dismutase, which comprises the steps of performing crude extraction on seeds or leaves of jatropha curcas by using a phosphate buffer solution, and purifying by thermal denaturation, ammonium sulfate fractional salting out, acetone precipitation and molecular sieve column chromatography in sequence; optimizes the technological parameters for extracting superoxide dismutase and can give consideration to the specific activity and recovery rate of the superoxide dismutase. Comparing the above-mentioned methods for extracting SOD from loquat seeds and Jatropha curcas seeds or leaves, it is found that SOD purified from different plants are very different in activity, purity and yield, and the principles of the purification techniques used in the process of purifying SOD are similar, but the sequence or specific parameters of the purification techniques are very different because basically all living bodies contain SOD, but they are different in structure, chemical characteristics and molecular weight, thus having different effects on the selection of the extraction method and the parameters of the extraction process. Further, since the crude enzyme solution extracted from different kinds of organisms has different impurities, the extraction conditions and the ease of extraction are different. It is important to find a plant with rich superoxide dismutase and easy extraction.
According to the invention, through research on different kinds of plants, the antioxidant system of the hyper-enriched plant is found to be stronger than that of a common plant, so that the use of the hyper-enriched plant for extracting SOD has advantages over the use of the common plant for extracting SOD. The super-enriched plant is a special engineering plant of a plant extraction technology, can be used for super-enriching heavy metal elements from soil and transporting the heavy metal elements to an overground part, and can be used for collecting the plant biomass waste material enriched with the heavy metals by mowing. At present, however, the mowing waste generated in the plant extraction technology is generally subjected to hazardous waste treatment after being reduced by a solidification and incineration technology in actual operation, and the incineration treatment not only increases the repair cost, but also wastes the physiologically active substances in the mowing waste. Therefore, a green treatment method which can effectively recycle the mowing biological waste generated in the plant extraction technology is very important under the condition that the farmland pollution problem in China is increasingly prominent. Therefore, the invention provides a method for extracting superoxide dismutase, which takes super-enriched plants as raw materials to extract the superoxide dismutase, not only can enrich a large amount of superoxide dismutase, but also can provide enough market demand for the mowing waste of the super-enriched plants to solve the resource problem.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for preparing superoxide dismutase enriched dry powder, which takes super-enriched plants as raw materials to extract the superoxide dismutase enriched dry powder, not only can enrich a large amount of superoxide dismutase and provide richer and more stable sources for the superoxide dismutase, but also can solve the problem of recycling the mowing biomass waste in the plant extraction technology.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing a superoxide dismutase enriched dry powder, said method comprising: after the super-enriched plant is pretreated, the super-enriched plant is subjected to the steps of leaching, salting out, impurity removal, concentration, precipitation enrichment and freeze drying, and the superoxide dismutase enriched dry powder is obtained.
Further, in the precipitation enrichment step, acetone is slowly added into the super-enriched plant treatment liquid to be precipitated and enriched until the volume concentration of the acetone in the solution reaches 60-70%, standing is carried out, and then the precipitate is obtained through centrifugation to remove the acetone. Preferably, the volume concentration of acetone in the solution is up to 65%. Further, the standing time is 0.1-24h, and the temperature is 10 ℃. Further, the precipitation enrichment is carried out at 4-30 ℃.
Further, in the precipitation and enrichment step, precipitation and impurity removal are carried out on the super-enriched plant treatment liquid to be precipitated and enriched, namely acetone is slowly added into the super-enriched plant treatment liquid to be precipitated and enriched until the volume concentration of the acetone in the solution reaches 30-55%, standing is carried out, and supernatant liquid is centrifugally collected for later use; preferably, the acetone is present in a concentration of up to 30% by volume. Further, the standing time is 0.1-5h, and the temperature is 10 ℃. Further, the precipitation and impurity removal are carried out at 4-30 ℃.
Further, in the precipitation enrichment step, the increase rate of the volume concentration of the acetone in the solution is increased by 0.1-2% per minute; preferably, it is 0.1% per minute.
Further, in the precipitation enrichment step, the centrifugation time is 10-120min, the temperature is 4-10 ℃, and the rotation speed is 4000-15000 rpm.
The two acetone extractions are used for enriching SOD, removing impurities and improving the SOD activity ratio of the final product; the supernatant is obtained by centrifugation after the first acetone addition, and the precipitate is obtained by centrifugation after the second acetone addition, because the SOD of the hyper-enriched plant does not precipitate when the acetone concentration is 30-55%, and precipitates when the acetone concentration is increased to 60-70%. Therefore, impurities can be better removed by reasonably controlling the addition amount of the acetone; the addition rate of acetone also has obvious influence on impurity removal, and the excessive high addition rate of acetone can cause the local concentration in the extracting solution to be too high, so that protein which is not in the target concentration is precipitated, the product quality is unstable, the impurity removal effect is poor, or SOD is separated out when SOD is not precipitated, and even partial SOD inactivation is caused. And the speed is too slow, so that the treatment time is prolonged, and the process cost is increased. Meanwhile, acetone is extremely easy to volatilize, is added too slowly for a long time, and can cause excessive volatilization of acetone, so that the final concentration is influenced, and the expected effect of the step cannot be perfectly achieved.
Further, the hyper-enrichment plant is selected from one or more of arsenic hyper-enrichment plant ciliate desert-grass, arsenic hyper-enrichment plant eriosema macrophylla, cadmium hyper-enrichment plant aizoon stonecrop, cadmium hyper-enrichment plant semimine stonecrop, manganese hyper-enrichment plant pokeberry, nickel hyper-enrichment plant Alyssum murale, and silver hyper-enrichment plant Indian mustard. Further, preparing the superoxide dismutase enriched dry powder by taking the mowing waste of the hyper-enriched plants as a raw material.
Further, in the step of pretreating the hyper-enriched plants, the mowing waste of the hyper-enriched plants is washed, freeze-dried until the water content is less than 10%, ground by a grinder and sieved. The purpose of freeze-drying is to better preserve the activity of SOD in the mown waste.
Further, in the leaching step, the pretreated hyper-enriched plant is immersed in a buffer solution containing a metal complexing agent, then homogenate is crushed, the resulting homogenate is filtered, leaching and filtering are repeated at least once, and filtrates are combined.
Further, in the leaching step, the heavy metal complexing agent is one of EDTA disodium, mercaptoethanol, ferric sulfate or cysteine. Preferably, the metal complexing agent is disodium EDTA.
Further, in the leaching step, the buffer solution is selected from one of phosphate buffer solution, Tris-HCl or glycinamide.
Further, in the leaching step, the buffer solution containing the metal complexing agent contains 0.005-0.05g of the metal complexing agent per 100mL of the buffer solution; preferably, the buffer containing the metal complexing agent contains 0.01g of the metal complexing agent per 100mL of the buffer.
Further, in the leaching step, the pH value of the buffer solution containing the metal complexing agent is 7-8.
Further, in the leaching step, the feed-liquid ratio of the mowing waste to the buffer solution containing the metal complexing agent is 1: 2-8 (w/v).
Further, in the leaching step, the temperature of the homogenate is 10-25 ℃.
In the leaching step, the pH value of the buffer solution containing the metal complexing agent and the dosage of the metal complexing agent are very important, the pH value is maintained to be alkalescent, the influence of a large amount of heavy metals contained in the mowing waste on the activity of the SOD enzyme is reduced, and the concentration of the metal complexing agent is controlled to avoid enzyme inactivation caused by complexing of metal ions carried by the SOD enzyme; the feed-liquid ratio is determined according to the water content of the smashed mowing waste, and the aim is to ensure that SOD in the mowing waste is extracted as much as possible.
Further, in the salting-out step, ammonium sulfate is added into the hyper-enriched plant treatment fluid to be salted out, and after standing, the mixture is centrifuged to obtain supernatant; preferably, in the salting-out step, ammonium sulfate is added to the filtrate to 5-20% saturation; more preferably, in the salting-out step, ammonium sulfate is added to the filtrate to 10% saturation.
Further, in the salting-out step, the standing time is 1-5h, and the standing temperature is 4-25 ℃.
In the salting-out step, low saturation is selected and is far lower than the prior art, because the salting-out in the prior art is the main step of removing the protein, while the invention mainly considers the whole process and mainly removes the extremely small suspended residues so as to protect the ultrafiltration equipment used in the subsequent steps, and the purification of the protein is only a side effect.
Further, in the impurity removal step, deionized water is added into the super-enriched plant treatment liquid to be subjected to impurity removal for dilution, and ultrafiltration is carried out until the filtrate is clear and colorless, and trapped fluid is taken; preferably, the ultrafiltration has a molecular weight cut-off of 3-10 kDa.
Further, in the impurity removal step, the addition amount of the deionized water is 0-5 times of that of the supernatant. Deionized water is added to accelerate the ultrafiltration speed, and whether the deionized water is added or not and the addition amount of the deionized water can be selected according to actual conditions.
Further, in the impurity removal step, the molecular weight cut-off of ultrafiltration is 3-10 kDa. This step can remove heavy metals, some hetero-proteins, some polysaccharides, some polypeptides, salts and other small molecule compounds.
Further, in the concentration step, the ultra-filtration concentration is performed on the ultra-enriched plant treatment liquid to be concentrated
Further, in the concentration step, the ultrafiltration has a molecular weight cut-off of 3-10 kDa.
Further, in the concentration step, the volume of the concentrated solution is 0.03-0.1 times of that of the trapped solution.
The main purpose of the concentration step is to greatly reduce the usage amount of organic solvent in the subsequent steps, reduce the cost, reduce the pollution and save the process time.
Further, before the precipitation enrichment step, the method also comprises a step of carrying out heat shock on the super-enriched plant treatment fluid to be precipitated and enriched, namely, heating the super-enriched plant treatment fluid to be precipitated and enriched at 60-80 ℃ for 20-120min, and then rapidly cooling to 4-10 ℃. The heat shock step may further remove contaminating proteins.
Further, freeze drying the precipitate enriched with superoxide dismutase obtained in the precipitate enrichment step to prepare the superoxide dismutase enriched dry powder.
Further, in the step of freeze drying, the precipitate is placed under the condition of negative pressure or low-temperature drying at the temperature of-20-10 ℃ until no smell can be detected; dissolving the precipitate in 50-150 times of water (w/v), and freeze drying to constant weight to obtain superoxide dismutase enriched dry powder. In the step, the acetone is removed mainly by utilizing the characteristic of volatilizable acetone, so that the quality of the product is ensured.
At present, the types of plants which can be used for extracting superoxide dismutase and are reported in the literature are few, corn, garlic, soybean, lithospermum seeds and the like are mainly used, and the extraction method has no general application because the superoxide dismutase (SOD) contained in different organisms has certain differences in structure, chemical characteristics and molecular weight; the invention finds that the anti-oxidation system of the mowing waste of the super-enriched plants is more powerful than that of the common plants, the super-enriched plant extraction is more advantageous than that of the common plants, and according to the characteristics of the plants, a method for preparing superoxide dismutase enriched dry powder from the mowing waste of typical super-enriched plants is obtained through a large amount of experimental research and creative labor, and the method is applicable to various super-enriched plants.
The preparation method comprises the steps of pretreating raw materials, and then leaching, wherein a buffer solution containing a metal complexing agent is adopted for leaching, because the mowing waste of the hyper-enriched plants contains a large amount of heavy metals, the heavy metals have great influence on the activity of SOD, and the buffer solution containing the metal complexing agent reduces the activity loss caused by excessive heavy metals; salting out ammonium sulfate, adsorbing the tiny plant residues remained in the extracting solution through the separated protein, further improving the clarity of the solution, and simultaneously protecting ultrafiltration equipment; removing impurities by ultrafiltration to remove heavy metals, impure proteins and other salts; the concentration step is considered in terms of process cost and safety, is to reduce the volume of trapped fluid and reduce the dosage of the medicament in the subsequent purification step, is very important for amplification and industrialization, and is a necessary technical characteristic in the technical scheme of the invention, for example, if the concentration treatment is not carried out before the acetone treatment, the required acetone dosage is very large, the cost is increased, and the acetone is flammable and toxic and threatens the production safety, so the smaller the dosage is, the better the dosage is; in the precipitation enrichment step, the SOD protein is enriched by acetone fractional precipitation; finally removing the organic solvent, and freeze-drying to obtain the enriched dry powder of the superoxide dismutase.
The technical scheme of the invention has the following advantages:
(1) the invention provides a method for preparing superoxide dismutase enriched dry powder, which takes a super-enriched plant as a raw material to extract superoxide dismutase. According to the invention, through research on different kinds of plants, the super-enriched plant for treating heavy metal soil pollution is found to have a stronger antioxidant system than that of a common plant, so that the super-enriched plant is more advantageous in extracting SOD than that of a common plant, the super-enriched plant is used as a raw material to extract superoxide dismutase, so that a large amount of superoxide dismutase can be enriched, a richer and more stable source is provided for the superoxide dismutase, the problem of recycling the mowing biomass waste in the plant extraction technology can be solved, the super-enriched plant mowing waste can be recycled, the mowing biomass waste in the plant extraction technology is changed into things of value by using the method, the current waste situation that the existing mowing biomass waste is subjected to incineration reduction is changed, and the method is suitable for various typical super-enriched plants, such as: ciliate desert-grass, sedum aizoon and sedum alfredii hance.
(2) The invention provides a method for preparing superoxide dismutase enriched dry powder, which comprises the following steps: after the hyper-enriched plant is pretreated, the enriched dry powder of the superoxide dismutase is obtained by the steps of leaching, salting out, impurity removal, concentration, precipitation enrichment and freeze drying. The method is suitable for preparing SOD enriched dry powder by cutting waste materials of a large class of plants, namely super-enriched plants, the selection of technical means in each step and the screening of parameters in each technical means are suitable for most commonly used super-enriched plants after test fine selection, and all the steps are mutually related, have a synergistic effect and cannot be simply replaced or randomly deleted. In short, the extraction conditions of different plants are different, but the method and conditions found out by the inventor are applicable to typical hyper-enriched plants such as ciliate desert-grass, sedum alfredii hance, sedum aizoon, sedum plumbizincicola and the like.
(3) In the step of precipitation and enrichment, acetone is slowly added into the super-enriched plant treatment liquid to be precipitated and enriched until the volume concentration of the acetone in the solution reaches 60-70%, standing is carried out, and then the precipitate is centrifugally taken to remove the acetone. By selecting the acetone with the concentration for precipitation and enrichment, the superoxide dismutase in the super-enriched plants can be precipitated, the superoxide dismutase is greatly enriched, and the activity ratio of the final product is improved.
(4) The invention provides a method for enriching superoxide dismutase, which comprises the steps of carrying out precipitation and impurity removal on a super-enriched plant treatment solution to be precipitated and enriched, namely slowly adding acetone into the super-enriched plant treatment solution to be precipitated and enriched until the volume concentration of the acetone in the solution reaches 20-40%, standing, centrifuging and collecting supernatant for later use; through the steps, the superoxide dismutase of the hyper-enriched plant can be further enriched and purified, and the activity ratio of the final product is further improved.
(5) According to the method for extracting superoxide dismutase, the increase rate of the volume concentration of acetone in the solution is 0.1-2% per minute; preferably, it is 0.1% per minute. By controlling the increase rate of the concentration of acetone, namely controlling the addition rate of acetone, the method avoids the problem that the addition rate of acetone is too high, so that the partial concentration of acetone in the extracting solution is too high, and protein which cannot be precipitated under the target concentration is precipitated, so that the product quality is unstable, the impurity removal effect is poor, or SOD is separated out when SOD is not precipitated, and even partial SOD inactivation is caused; meanwhile, the problem that the speed is too slow, the treatment time is prolonged, the process cost is increased, and meanwhile, acetone is extremely easy to volatilize, is added too slowly and is too long, so that excessive volatilization of acetone can be caused, the final concentration is influenced, and the expected effect of the step cannot be perfectly achieved.
Detailed Description
The embodiments of the present invention are illustrated below by specific examples, and unless otherwise indicated, the experimental methods disclosed in the present invention are performed by using conventional techniques in the art, and reagents and raw materials used in the examples are commercially available.
Example 1
Preparing superoxide dismutase enriched dry powder from cutting waste materials of cadmium hyper-enrichment plant sedum aizoon (1), taking 10kg of overground part of cadmium enriched sedum aizoon (Hylotelehium spectabile) which is freshly cut for one hour, cleaning by tap water, removing impurities such as floating dust and the like, and drying; freeze drying for 72 hr until the water content is less than 10%, pulverizing at 20 deg.C, and sieving with 100 mesh sieve.
(2) Soaking the raw materials in 70L phosphate buffer solution (containing 7g disodium ethylene diamine tetraacetate) with pH of 7.6, and pulverizing at high speed at 20 deg.C to obtain homogenate; filtering the homogenate by a 200-mesh double-layer nylon filter cloth, repeatedly leaching the homogenate and filtering for three times under the same condition, and combining the filtrates.
(3) Ammonium sulfate was added to the filtrate to 10% saturation, and after standing at 10 ℃ for 1 hour, the precipitate was removed by centrifugation at 4000rpm at 10 ℃ until the solution was clear, yielding 180L of supernatant.
(4) Adding 3 times volume of deionized water into the supernatant for dilution, performing ultrafiltration with 3kDa until the filtrate is clear and colorless, and taking trapped fluid.
(5) Concentrating the obtained retentate by 3kDa ultrafiltration to obtain concentrated solution 9L; heating the obtained concentrated solution at 70 deg.C for 40min, and rapidly cooling to 4 deg.C.
(6) Keeping the concentrated solution at 4 ℃, stirring the concentrated solution, slowly adding acetone, wherein the volume concentration of the acetone is increased at a rate of 0.1%/min until the concentration of the acetone in the solution reaches 30%, standing the solution at 4 ℃ for 0.1 hour, centrifuging the solution at 10 ℃ and 10000rpm for 30 minutes, and collecting supernatant; stirring the supernatant at 4 ℃ and slowly adding acetone at the rate of 0.1%/min until the acetone concentration of the solution is increased from 30% to 60%, and standing at 10 ℃ for 1 hour; centrifuging at 10000rpm at 4 deg.C for 30min, collecting precipitate, repeating the above centrifuging steps for the supernatant twice, collecting precipitate, and mixing the three centrifugations.
(7) Placing the precipitate at 10 deg.C under vacuum condition, removing acetone until no smell can be detected; dissolving the precipitate in 600mL of ultrapure water, and freeze-drying until the weight is constant to obtain the superoxide dismutase enriched dry powder.
The experiment proves that the mass of the rhodiola sachalinensis superoxide dismutase enriched dry powder is 4.0g, and the specific activity is 140U/mg.
Example 2
Preparation of superoxide dismutase enriched dry powder from cutting waste material of arsenic super-enriched plant ciliate desert-grass
(1)100kg of fresh centipede grass (Pteris vitata) aerial parts are harvested within half an hour, feather leaves are collected and washed by distilled water, impurities such as floating dust and the like are removed, and the feather leaves are dried.
(2) Soaking the raw materials in 400L phosphate buffer solution (containing 40g disodium ethylene diamine tetraacetate) with pH 7.8, and pulverizing at 15 deg.C and homogenizing at high speed; filtering the homogenate liquid with 200-mesh nylon filter cloth, repeatedly leaching the homogenate liquid and filtering for three times, and combining to obtain 1000L of filtrate.
(3) Adding ammonium sulfate into the filtrate to reach saturation of 10%, standing at 4 deg.C for 2 hr, centrifuging at 4000rpm at 4 deg.C until the supernatant is clear, and collecting the supernatant.
(4) Diluting the supernatant with 3 times volume of deionized water, ultrafiltering with 10kDa until the ultrafiltrate is clear and colorless, and collecting the trapped fluid.
(5) The retentate 10kDa was concentrated to 0.03 times the original volume. The concentrated solution is heated for 30 minutes at 70 ℃, and then is rapidly cooled to 4 ℃.
(6) Keeping the temperature at 4 ℃, stirring the concentrated solution, slowly adding acetone, increasing the acetone concentration by 0.9 percent per minute until the acetone concentration of the solution reaches 50 percent, and standing for 1 hour at 4 ℃; centrifuging at 15000rpm at 4 deg.C for 20min, and collecting supernatant; stirring the supernatant at 4 ℃ and slowly adding acetone, wherein the increase rate of the acetone concentration is 1% per minute until the acetone concentration of the solution reaches 60%, standing the solution at 4 ℃ for 24 hours, centrifuging the solution at 4 ℃ at 15000rpm for 20 minutes, taking the precipitate, repeating the centrifugation conditions of the supernatant twice, and combining the centrifugation precipitates for three times.
(7) And (3) placing the precipitate at a temperature of 20 ℃ below zero under negative pressure to remove acetone until no smell can be detected, dissolving the precipitate in 15 times of water (the relationship between the precipitate and the water is w/v, the unit is g/ml, the same applies below), and freeze-drying to constant weight to obtain the superoxide dismutase enriched dry powder.
The experiment proves that the mass of the ciliate desert-grass superoxide dismutase enriched dry powder is 59g, and the specific activity is 200U/mg.
Example 3
Preparation of superoxide dismutase enriched dry powder from cutting waste material of nickel super enriched plant Alyssum corsicum
(1)40kg of biomass waste on the overground part of a nickel hyper-enriched plant Alyssum coresicum which is freshly mown within 24 hours, cleaning the whole plant by using water, removing impurities such as floating dust and the like, and drying the whole plant by spinning; freeze drying to constant weight; the raw materials are ground into powder by a grinder after being cut up and sieved by a 100-mesh filter screen.
(2) Soaking the raw materials in 160L phosphate buffer solution (0.01 g disodium edetate per 100 mL) with pH 7, and pulverizing at high speed at 10 deg.C to obtain homogenate; filtering the homogenate with 200-mesh nylon filter cloth, repeatedly leaching the homogenate and filtering for three times, and combining the filtrates.
(3) Ammonium sulfate was added to the filtrate to 10% saturation, and after standing at 20 ℃ for 1 hour, the supernatant was centrifuged.
(4) Diluting the supernatant with 3 times volume of deionized water, ultrafiltering with 3kDa until the filtrate is clear and colorless, and collecting the trapped fluid.
(5) And (5) carrying out ultrafiltration concentration on the trapped fluid with 3kDa to obtain 10L concentrated solution. The concentrated solution is heated for 20 minutes at 80 ℃ and then rapidly cooled to 4 ℃.
(6) Keeping the temperature at 25 ℃, stirring the concentrated solution, slowly adding 5.38L of acetone, wherein the volume concentration of the acetone is increased by 0.1% per minute until the concentration of the acetone in the solution reaches 35%, standing for 0.4 hour at 4 ℃, centrifuging for 22 minutes at 13000rpm at 10 ℃, and collecting the supernatant; stirring the supernatant at 25 deg.C, slowly adding 13.2L of acetone with the acetone concentration increasing rate of 0.5% per minute until the acetone concentration of the solution reaches 65%, standing at 8 deg.C for 3 hr, centrifuging at 13000rpm at 7 deg.C for 18 min, collecting precipitate, repeating the supernatant centrifuging conditions twice, and combining the three centrifugations.
(7) Placing the precipitate at 0 deg.C, drying at low temperature, and removing acetone until no smell can be detected; dissolving the precipitate in 2.38L water, and freeze drying to constant weight to obtain superoxide dismutase enriched dry powder.
The experiment shows that the specific activity of the Alyssum coresium superoxide dismutase enriched dry powder is 120U/mg, and the mass of the Alyssum coresium superoxide dismutase enriched dry powder is 20 g.
Example 4
Preparation of superoxide dismutase enriched dry powder from cutting waste material of arsenic super-enriched plant ciliate desert-grass and cadmium eight-treasure sedum
(1) 30kg of arsenic-enriched ciliate desert-grass and 20kg of cadmium-enriched sedum octandra biomass waste within 6 hours of fresh mowing, cleaning with water, spin-drying, and chopping.
(2) Cutting the raw materials, soaking in 200L phosphate buffer solution (containing 20g disodium ethylene diamine tetraacetate) with pH 7.8, and pulverizing at high speed at 10 deg.C to obtain homogenate; filtering the homogenate with 200-mesh nylon filter cloth, repeatedly leaching the homogenate and filtering for three times, and combining the filtrates to obtain 480L.
(3) To the filtrate was added 26.88kg of ammonium sulfate, and after standing at 4 ℃ for 1 hour, the supernatant was centrifuged.
(4) Diluting the supernatant with 1 volume of deionized water, repeatedly ultrafiltering at 10kDa until the filtrate is clear and colorless, and collecting the trapped fluid.
(5) The retentate was concentrated by 10kDa ultrafiltration to give 30L of concentrate. Uniformly heating the concentrated solution at 70 deg.C for 30min, and rapidly cooling to 4 deg.C.
(6) Stirring the concentrated solution at 10 deg.C, slowly adding 24.54L acetone with the acetone concentration increasing rate of 0.15% per minute until the acetone concentration reaches 45%, standing at 4 deg.C for 3 hr, centrifuging at 4 deg.C at 15000rpm for 20min, and collecting supernatant; stirring the supernatant at 10 ℃ and slowly adding acetone at the rate of increasing the acetone concentration by 0.1% per minute until the acetone concentration of the solution reaches 65%, standing for 3 hours at 4 ℃, centrifuging for 30 minutes at 13000rpm at 4 ℃, taking the precipitate, repeating the centrifugation conditions of the supernatant twice, and combining the centrifugation precipitates for three times.
(7) Placing the precipitate at-20 deg.C, drying at low temperature to remove acetone until no smell can be detected; dissolving the precipitate in 100 times of water (w/v), and freeze-drying until the weight is constant to obtain the superoxide dismutase enriched dry powder.
The experiment shows that the mass of the ciliate desert-grass and sedum sarmentosum mixed superoxide dismutase enriched dry powder is 25g, and the specific activity is 175U/mg.
Example 5
Preparing superoxide dismutase enriched dry powder (1) from the cutting waste of the cadmium-enriched Sedum hemimelaleum, cleaning the overground part of the cadmium-enriched Sedum hemimelaleum with distilled water, removing impurities such as floating dust and the like, and drying the overground part of the cadmium-enriched Sedum hemimelaleum within 20kg of fresh cutting half an hour.
(2) Soaking the raw materials in 100L Tris-HCl buffer solution (containing 10g cysteine) with pH 7.4, and pulverizing at high speed at 20 deg.C to obtain homogenate; filtering the homogenate by a nylon filter cloth with 200 meshes, repeatedly leaching the homogenate and filtering for three times, and combining to obtain filtrate 301L.
(3) Adding ammonium sulfate into the filtrate to saturation degree of 12%, standing at 4 deg.C for 2 hr, centrifuging at 6000rpm and 4 deg.C until the supernatant is clear, and collecting the supernatant.
(4) Diluting the supernatant with 3 times volume of deionized water, ultrafiltering with 3kDa until the ultrafiltrate is clear and colorless, and collecting the trapped fluid.
(5) The retentate 3kDa was concentrated to 0.03 times the original volume. The concentrated solution is heated for 30 minutes at 70 ℃, and then is rapidly cooled to 7 ℃.
(6) Keeping the temperature at 7 ℃, stirring the concentrated solution, slowly adding acetone, wherein the increase rate of the acetone concentration is 0.7 percent per minute until the acetone concentration of the solution reaches 50 percent, and standing for 1 hour at 4 ℃; centrifuging at 15000rpm at 4 deg.C for 20min, and collecting supernatant; stirring the supernatant at 4 ℃ and slowly adding acetone at the rate of acetone concentration increase of 0.7% per minute until the acetone concentration of the solution reaches 62%, standing for 2 hours at 4 ℃, centrifuging at 4 ℃ for 20 minutes at 5000rpm, taking the precipitate, repeating the centrifugation conditions of the supernatant twice, and combining the centrifugation precipitates for three times.
(7) Placing the precipitate at-20 deg.C to allow acetone to naturally volatilize until no smell can be detected, dissolving the precipitate in 150 times of water (w/v), and freeze drying to constant weight to obtain superoxide dismutase enriched dry powder.
The experiment shows that the quality of the rhodiola hemiminea superoxide dismutase enriched dry powder is 12g, and the specific activity is 160U/mg.
Example 6
Preparation of superoxide dismutase enriched dry powder from manganese super-enriched plant phytolacca acinosa cutting waste material
(1)50kg of manganese-rich pokeberry leaves which are freshly mown for half an hour are cleaned by distilled water, impurities such as floating dust are removed, and the pokeberry leaves are dried.
(2) Soaking the raw materials in 200L glycinamide buffer solution (containing 10g mercaptoethanol) with pH 7.8, and pulverizing at 20 deg.C and high speed to obtain homogenate; filtering the homogenate with 200-mesh nylon filter cloth, repeatedly leaching the homogenate and filtering for three times, and combining to obtain filtrate 601L.
(3) Adding ammonium sulfate into the filtrate to saturation degree of 15%, standing at 4 deg.C for 2 hr, centrifuging at 7000rpm and 4 deg.C until the supernatant is clear, and collecting the supernatant.
(4) Diluting the supernatant with 5 times volume of deionized water, ultrafiltering with 10kDa until the ultrafiltrate is clear and colorless, and collecting the trapped fluid.
(5) The retentate 3kDa was concentrated to 0.03 times the original volume. The concentrated solution is heated for 30 minutes at 70 ℃, and then is rapidly cooled to 7 ℃.
(6) Keeping the temperature at 7 ℃, stirring the concentrated solution, slowly adding acetone, increasing the acetone concentration by 0.1 percent per minute until the acetone concentration of the solution reaches 50 percent, and standing for 1 hour at 4 ℃; centrifuging at 5000rpm at 4 deg.C for 20min, and collecting supernatant; stirring the supernatant at 4 deg.C, slowly adding acetone at an acetone concentration increasing rate of 0.8% per minute until the acetone concentration of the solution reaches 64%, standing at 4 deg.C for 1 hr, centrifuging at 4 deg.C at 4800rpm for 20min, collecting precipitate, repeating the above centrifuging conditions for two times, and mixing the three centrifugations.
(7) And placing the precipitate at 10 ℃ to naturally volatilize the acetone until no smell can be detected, dissolving the precipitate in 150 times of water (w/v), and freeze-drying to constant weight to obtain the superoxide dismutase enriched dry powder.
The experiment proves that the mass of the pokeberry root superoxide dismutase enriched dry powder is 20g, and the specific activity is 120U/mg.
Example 7
Preparation of superoxide dismutase enriched dry powder from cutting waste material of cadmium super-enriched plant Sedum alfredii Hance and Sedum hemimineum Hance
(1)25kg of fresh sedum alfredii and 40kg of overground part of sedum alfredii within half an hour of mowing, cleaning with distilled water, removing impurities such as floating dust and the like, and spin-drying.
(2) Soaking the raw materials in 260L phosphate buffer solution (containing 10g mercaptoethanol) with pH 7.2, and pulverizing at 20 deg.C and high speed to obtain homogenate; filtering the homogenate by a 200-mesh nylon filter cloth, repeatedly leaching the homogenate and filtering for three times, and combining to obtain 780L filtrate.
(3) Adding ammonium sulfate into the filtrate to saturation degree of 10%, standing at 4 deg.C for 2 hr, centrifuging at 4500rpm at 4 deg.C until the supernatant is clear, and collecting the supernatant.
(4) Diluting the supernatant with 2 times volume of deionized water, performing ultrafiltration with 10kDa until the ultrafiltrate is clear and colorless, and collecting the trapped fluid.
(5) The retentate 3kDa was concentrated to 0.1 times the original volume. The concentrated solution is heated for 30 minutes at 70 ℃, and then is rapidly cooled to 7 ℃.
(6) Keeping the temperature at 7 ℃, stirring the concentrated solution, slowly adding acetone, wherein the increase rate of the acetone concentration is 0.4 percent per minute until the acetone concentration of the solution reaches 50 percent, and standing for 1 hour at 4 ℃; centrifuging at 5000rpm at 4 deg.C for 20min, and collecting supernatant; stirring the supernatant at 4 deg.C, slowly adding acetone at an acetone concentration increasing rate of 0.11% per minute until the acetone concentration of the solution reaches 64%, standing at 4 deg.C for 1.5 hr, centrifuging at 4 deg.C at 4800rpm for 20min, collecting precipitate, repeating the centrifuging conditions of the supernatant twice, and mixing the three centrifugations.
(7) And placing the precipitate at 10 ℃ to naturally volatilize the acetone until no smell can be detected, dissolving the precipitate in 150 times of water (w/v), and freeze-drying to constant weight to obtain the superoxide dismutase enriched dry powder.
The experiment proves that the mass of the mixed superoxide dismutase enriched dry powder of the sedum alfredii hance and the sedum hemimineum is 40g, and the specific activity is 70U/mg.
Example 8
Preparation of superoxide dismutase enriched dry powder from cadmium hyper-enriched plant sedum alfredii hance and manganese hyper-enriched plant phytolacca acinosa cutting waste material
(1)40kg of fresh sedum alfredii and 40kg of overground part of phytolacca americana which are full of cadmium and are cut for half an hour are cleaned by distilled water, impurities such as floating dust are removed, and the parts are dried.
(2) Soaking the raw materials in 240L Tris-HCl buffer solution (containing 10g mercaptoethanol) with pH 7.7, and pulverizing at high speed at 25 deg.C to obtain homogenate; filtering the homogenate by a 200-mesh nylon filter cloth, repeatedly leaching the homogenate and filtering for three times, and combining to obtain 780L filtrate.
(3) Adding ammonium sulfate into the filtrate to saturation degree of 5%, standing at 4 deg.C for 4 hr, centrifuging at 4500rpm at 4 deg.C until the supernatant is clear, and collecting the supernatant.
(4) And (5) carrying out ultrafiltration on the supernatant with 8kDa until the ultrafiltrate is clear and colorless, and taking trapped fluid.
(5) The retentate 5kDa was concentrated to 0.1 times the original volume.
(6) Stirring the concentrated solution at 15 ℃, slowly adding acetone, increasing the acetone concentration by 2% per minute until the acetone concentration of the solution reaches 55%, and standing for 3 hours at 4 ℃; centrifuging at 5000rpm for 90 min at 4 deg.C, and collecting supernatant; stirring the supernatant at 4 deg.C, slowly adding acetone at an acetone concentration increasing rate of 0.2% per minute until the acetone concentration of the solution reaches 70%, standing at 4 deg.C for 1.5 hr, centrifuging at 4 deg.C at 4800rpm for 70 min, collecting precipitate, repeating the centrifuging conditions of the supernatant twice, and mixing the three centrifugations.
(7) And placing the precipitate at 5 ℃ to naturally volatilize acetone until no smell can be detected, dissolving the precipitate in 100 times of water (w/v), and freeze-drying to constant weight to obtain the superoxide dismutase enriched dry powder.
The experiment shows that the mass of the mixed superoxide dismutase enriched dry powder of the sedum alfredii hance and the pokeberry is 46g, and the specific activity is 110U/mg.
Comparative example 1
This comparative example is substantially the same as example 1, except that step (6) in example 1 was adjusted, the other step sequence parameters were not changed, and step (6) was adjusted to: keeping the concentrated solution at 4 ℃, stirring the concentrated solution, slowly adding acetone, wherein the volume concentration of the acetone is increased at a rate of 0.1%/min until the concentration of the acetone in the solution reaches 25%, standing the solution at 4 ℃ for 0.1 hour, centrifuging the solution at 10 ℃ and 10000rpm for 30 minutes, and collecting supernatant; stirring the supernatant at 4 ℃ and slowly adding acetone at the rate of 0.1% per minute until the acetone concentration of the solution is increased from 25% to 50% and then standing for 1 hour at 10 ℃; centrifuging at 10000rpm at 4 deg.C for 30min, collecting precipitate, repeating the above centrifuging steps for the supernatant twice, collecting precipitate, and mixing the three centrifugations.
The experiment proves that the final product has the mass of 4g and the specific activity of 4U/mg.
Comparative example 2
The step (6) in the embodiment 1 is adjusted, the sequence parameters of other steps are not changed, and the step (6) is adjusted as follows: keeping the concentrated solution at 4 ℃, stirring the concentrated solution, slowly adding acetone, wherein the volume concentration of the acetone is increased at a rate of 0.1%/min until the concentration of the acetone in the solution reaches 30%, standing the solution at 4 ℃ for 0.1 hour, centrifuging the solution at 10 ℃ and 10000rpm for 30 minutes, and collecting supernatant; stirring the supernatant at 4 ℃ and slowly adding acetone at the rate of 0.1% per minute until the acetone concentration of the solution is increased from 30% to 80% and then standing for 1 hour at 10 ℃; centrifuging at 10000rpm at 4 deg.C for 30min, collecting precipitate, repeating the above centrifuging steps for the supernatant twice, collecting precipitate, and mixing the three centrifugations.
The experiment proves that the quality of the rhodiola sachalinensis superoxide dismutase enriched dry powder is 7.1g, and the specific activity is 75U/mg.
Comparative example 3
The step (6) in the embodiment 1 is adjusted, the sequence parameters of other steps are not changed, and the step (6) is adjusted as follows: keeping the concentrated solution at 4 ℃, stirring the concentrated solution, slowly adding acetone, wherein the volume concentration of the acetone is increased at a rate of 0.1%/min until the concentration of the acetone in the solution reaches 20%, standing the solution at 4 ℃ for 0.1 hour, centrifuging the solution at 10 ℃ and 10000rpm for 30 minutes, and collecting supernatant; stirring the supernatant at 4 ℃ and slowly adding acetone at the rate of 0.1% per minute until the acetone concentration of the solution is increased from 20% to 80% and then standing for 1 hour at 10 ℃; centrifuging at 10000rpm at 4 deg.C for 30min, collecting precipitate, repeating the above centrifuging steps for the supernatant twice, collecting precipitate, and mixing the three centrifugations.
The experiment proves that the quality of the rhodiola sachalinensis superoxide dismutase enriched dry powder is 9.2g, and the specific activity is 55U/mg.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (5)
1. A method for preparing superoxide dismutase enriched dry powder, which comprises the following steps: after the super-enriched plant is pretreated, the super-enriched plant is treated by the steps of leaching, salting out, impurity removal, concentration, precipitation enrichment and freeze drying, and the super-oxide dismutase enriched dry powder is obtained;
in the salting-out step, ammonium sulfate is added into the hyper-enriched plant treatment fluid to be salted out to 5-20% saturation, and the mixture is stood and centrifuged to take supernatant;
in the impurity removal step, deionized water is added into the supernatant for dilution, and ultrafiltration is carried out until the filtrate is clear and colorless, and trapped fluid is taken;
in the concentration step, the trapped fluid is subjected to ultrafiltration concentration; the volume of the concentrated solution is 0.03-0.1 times of the trapped fluid; heating the concentrated solution at 60-80 deg.C for 20-120min, and rapidly cooling to 4-10 deg.C;
in the precipitation enrichment step, slowly adding acetone into the concentrated solution until the volume concentration of the acetone in the solution reaches 30-55%, standing, centrifuging, and collecting the supernatant for later use; the increase rate of the volume concentration of the acetone in the solution is 0.1 to 2 percent per minute;
in the precipitation enrichment step, slowly adding acetone into the supernatant until the volume concentration of the acetone in the solution reaches 60-70%, standing, centrifuging to obtain precipitate, and removing the acetone;
in the step of freeze drying, the precipitate which is obtained in the step of precipitate enrichment and is enriched with superoxide dismutase is freeze dried to prepare superoxide dismutase enriched dry powder;
the hyper-enrichment plant is selected from one of cadmium hyper-enrichment plant Sedum Palustre Kirilowii, cadmium hyper-enrichment plant Sedum hemimineum, arsenic hyper-enrichment plant Pteris vittata, nickel hyper-enrichment plant Alyssum murale and manganese hyper-enrichment plant Phytolacca acinosa.
2. The method for producing a superoxide dismutase rich dry powder as claimed in claim 1, wherein in the precipitation enrichment step, the increase rate of the acetone volume concentration in the solution is 0.1% per minute.
3. The method for producing a superoxide dismutase rich dry powder as claimed in claim 1, wherein in the leaching step, the pretreated super-rich plant is immersed in a buffer solution containing a metal complexing agent, then a homogenate is crushed, the resulting homogenate is filtered, leaching and filtering are repeated at least once, and the filtrates are combined.
4. The method for producing a superoxide dismutase rich dry powder as claimed in claim 3, wherein the metal complexing agent is one selected from the group consisting of disodium EDTA, mercaptoethanol, ferric sulfate and cysteine; the buffer solution is selected from one of phosphate buffer solution, Tris-HCl or glycinamide.
5. The method for producing a superoxide dismutase rich dry powder as claimed in claim 1, wherein in the step of removing impurities, the molecular weight cut off by ultrafiltration is 3 to 10 kDa.
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| 从大蒜细胞中分离纯化出超氧化物歧化酶;邓旭 等;《食品科学》;20011231;第22卷(第9期);47-49 * |
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